Methods and compounds for the treatment or prevention of hypercytokinemia and severe influenza

ABSTRACT

The invention provides a p38 MAPK inhibitor of Formula I, or a pharmaceutically acceptable salt or solvate thereof: Formula I for use in the treatment or prevention of hypercytokinemia in a human patient; wherein R is C 1-3 alkyl, optionally substituted by one or more halo, NR 1 R 2  or hydroxy, and R 1  and R 2  are independently H, halo or C 1-3 alkyl, optionally substituted by one or more F. Also provided are compositions for use in the treatment or prevention of hypercytokinemia comprising the p38 MAPK inhibitor of Formula I; and methods for treating or preventing hypercytokinemia in a human patient in need thereof comprising administering to the patient a therapeutically or prophylactically effective amount of a p38 MAPK inhibitor of Formula I. The invention also provides a p38 MAPK inhibitor and an antimicrobial agent, such as an antiviral agent, for use in the treatment or prevention of hypercytokinemia.

FIELD OF THE INVENTION

The present invention relates to methods and compounds for the treatmentor prevention of hypercytokinemia and, in particular, hypercytokinemiaassociated with severe influenza.

The invention also provides pharmaceutical compositions for thetreatment or prevention of hypercytokinemia and hypercytokinemiaassociated with severe influenza.

BACKGROUND TO THE INVENTION

Hypercytokinemia is defined as a sudden surge in the circulating levelsof pro-inflammatory cytokines, such as IL-1, IL-6 and TNF (Croft, M.,The role of TNF superfamily members in T-cell function and diseases, NatRev Immunol, 2009; 9: 271-85).

Hypercytokinemia, or “cytokine storm”, or “cytokine cascade”, isassociated with various conditions including infectious diseases,non-infectious diseases, autoimmune reactions and adverse drugreactions. Inflammation is part of the complex biological response ofbody tissues to harmful stimuli, such as viruses, damaged cells, orirritants. It is a protective response that involves the immune system,blood vessels and numerous proteins. The purpose of inflammation is toeliminate the initial cause of cell injury, to clear out dead and dyingcells and to initiate tissue repair. In normal circumstancesinflammation is short lived and repair to damaged tissues normallystarts 2 to 3 days after the onset of symptoms.

Hypercytokinemia develops when the inflammatory response is exaggeratedand high levels of pro-inflammatory proteins (cytokines) are releasedwhich can lead to damage of the blood vessels in the lung and othertissues resulting in liquid leaking into tissues (oedema). Rather thanbeing protective the inflammatory response becomes destructive. Thisexaggerated inflammatory response can be viewed as a non-linear processwhere there is a critical point—a phase transition, tipping point, orthreshold—where the normal inflammatory response becomes an abnormalresponse.

A variety of anti-inflammatory drugs and adjunct approaches have beenadopted in an attempt to target hypercytokinemia. These includetreatment with corticosteroids, aspirin, monoclonal antibodies (MAbs),anti-cytokine and anti-chemokine agents, plasma exchange, and statins.Despite these efforts, none of these approaches have proved to beeffective, and some have worsened the outcome (Brun-Buisson et al.,Early corticosteroids in severe influenza A/H1N1 pneumonia and acuterespiratory distress syndrome, Am J Respir Crit Care Med. 2011 May1;183(9):1200-6). The therapeutic approaches of the present inventionare aimed at balancing the response by attenuating the inflammatoryresponse rather than ablating it, thus reducing the damaging effectscaused by the exaggerated inflammatory response whilst preserving thehost's innate protective response.

Hypercytokinemia is seen in severe infections with three major influenzaviruses: the pandemic 1918-19 Spanish H1N1 influenza; H5N1 avianinfluenza and; the pandemic H1N1 influenza of 2009. When compared tohuman H1N1, H5N1 viruses are more potent inducers of pro-inflammatorycytokines in primary human respiratory epithelial cells, and thishyperinduction of cytokines is likely to contribute to the diseaseseverity of H5N1. The exact mechanism of hypercytokinemia in influenzais unknown, but endothelial cells have been identified as centralregulators of “cytokine storm” (Teijaro J R et al., Endothelial Cellsare Central Orchestrators of Cytokine Amplification during InfluenzaVirus Infection, Cell, 2011; 146: 980-991).

Influenza occurs globally with an annual attack rate estimated at 5%-10%in adults and 20%-30% in children. Illnesses can result inhospitalization and death mainly among high-risk groups (the very young,elderly or chronically ill). Worldwide, these annual epidemics areestimated to result in about 3 to 5 million cases of severe illness, andabout 250,000 to 500,000 deaths. (See WHO factsheet number 211:https://web.archive.org/web/20160613040907/http://www.who.int/mediacentre/factsheets/fs211/en/).

In North America, seasonal influenza causes excess hospitalisations in230-1670 per 100,000 persons aged >65 years, 32,000respiratory/cardiovascular deaths and 43,000 all-cause deaths annually.Persons with chronic medical conditions (e.g. pulmonary, cardiovascular,liver, renal and neurological diseases, diabetes or immunosuppression)have a >30-fold increase in risk of hospitalisation and death.

Among the circulating seasonal influenza subtypes, H3N2 is usually amore frequent cause of severe illness and hospitalisation. (See Lee, N.et al., Outcomes of adults hospitalised with severe influenza, Thorax,2010; 65: 510-515).

The situation can be even worse during influenza pandemics. In early2009, a novel influenza A/H1N1 virus (pH1N1) emerged and rapidly causeda pandemic. It has been estimated that in some populations, up to 20-40%of individuals were affected and resulted in excessive hospitalizationsand deaths. In the United States, 195,000-403,000 people werehospitalized for severe pH1N1 infection and 8,870-18,300 died by April2010. The pH1N1 virus has continued to co-circulate with the seasonalinfluenza viruses in the community.

While most patients develop mild upper respiratory-tract infection withpH1N1, some progress to develop severe lower respiratory-tractcomplications, such as, for example, pneumonia, or transition toexperiencing symptoms that do not resolve or improve after several days(>2 days). In contrast to seasonal influenza, young adults andpreviously healthy individuals may also develop severe respiratorycomplications such as, for example, pneumonia and acute respiratorydistress syndrome (ARDS). Among hospitalized adults, between 9-34% mayrequire intensive care because of progressive respiratory failure, whichcan be associated with high mortality (14-46%); notably, some of themanifestations (e.g. pneumonia, ARDS, multi-organ failure) are quitesimilar to those of H5N1 avian influenza. (See Lee, N. et al., Cytokineresponse patterns in severe pandemic 2009 H1N1 and seasonal influenzaamong hospitalised adults, PloS One, 2011; 6: e26050).

WO 01/19322 A2 (SmithKline Beecham Corp) claims a method of treatinginfluenza-induced pneumonia which method comprises administering to thehuman an effective amount of a CB SP/p3 8 inhibitor.

The p38 MAP kinases comprise a mitogen-activated protein kinasesubfamily that regulates a variety of cellular processes including cellgrowth processes, cell differentiation, apoptosis and cellular responsesto inflammation. The p38 MAP kinases are regulated by cytokine receptorsand can be activated in response to bacterial or viral pathogens.

WO 2004/076450 A1 discloses the use of certain pyrazolopyridinederivatives for the treatment or prevention of diseases mediated byTNF-α, IL-1, IL-6 and/or IL-8, including immune, autoimmune andinflammatory diseases, cardiovascular diseases, infectious diseases,bone resorption disorders, neurodegenerative diseases, proliferativediseases and processes associated with the induction ofcyclooxygenase-2.

WO 02/059083 A2 (SmithKline Beecham Corp) claims the use of substituted2,4,8-trisubstituted-8H-pyrido[2,3-d]pyrimidin-7-one compounds andcompositions for treating a wide range of CBSP/p38 kinase mediateddiseases, including ARDS, chronic obstructive pulmonary disease andinfluenza-induced pneumonia.

U.S. 2011/003848 A1 (Butcher) discloses the use of polymorphic form ofthe p38 MAP kinase inhibitor,N-[3-tert-butyl-1-(3-chloro-4-hydroxyphenyl)-1H-pyrazol-5-yl]-N′-{2-[(3-{2-[(2-hydroxyethyl)sulfanyl]phenyl}[1,2,4]triazolo[4,3-a]pyridin-6-yl)sulfanyl]benzyl}urea, for treating obstructive,restrictive or inflammatory airways diseases of whatever type, etiologyor pathogenesis.

Meanwhile, WO 2015/173788 A1 (Westfälische Wilhelms-Universität Münster)claims a MEK inhibitor, p38 inhibitor and/or NF-κB inhibitor for use ina method for the prophylaxis and/or treatment of a co-infectioncomprising a bacterial infection and an influenza virus infection or abacterial infection alone.

A p38 MAPK inhibitor is disclosed in Mihara K, et al. A potent andselective p38 inhibitor protects against bone damage in murinecollagen-induced arthritis: a comparison with neutralization of mouseTNFalpha., Br. J. Pharmacol., 2008 (May);154(1):153-64, andGalan-Arriero I, et al. Early treatment with UR13870, a novel inhibitorof p38α mitogenous activated protein kinase, prevents hyperreflexia andanxiety behaviors, in the spared nerve injury model of neuropathic pain.Neurosci. Lett. 2015 (Sep) 14;604:69-74.

Generally, as discussed in more detail below, a number of differentanti-inflammatory agents have been proposed in the art for treatinginflammation associated with influenza infection.

The World Health Organisation (WHO) defines severe influenza as:Influenza in patients who present clinical and/or radiological signs oflower respiratory tract disease, central nervous system (CNS)involvement, severe dehydration or present secondary complications, suchas renal failure, multi-organ failure and septic shock (othercomplications can include rhabdomyolysis and myocarditis); influenzawhere there is exacerbation of underlying chronic disease; influenzawhere there is any other condition or clinical presentation thatrequires hospital admission for clinical management; and influenza wherethere are any of the following signs and symptoms of progressivedisease:

Symptoms and signs suggesting oxygen impairment or cardiopulmonaryinsufficiency: Shortness of breath (with activity or at rest),difficulty in breathing, tachypnoea, presence of cyanosis, bloody orcoloured sputum, chest pain, and low blood pressure; in children, fastor laboured breathing; and hypoxia, as indicated by pulse oximetry orarterial blood gases;

Symptoms and signs suggesting CNS complications: Altered mental status,unconsciousness, drowsiness, or difficult to awaken and recurring orpersistent convulsions (seizures), confusion, severe weakness, orparalysis;

Evidence of sustained or spreading virus replication or invasivesecondary bacterial infection based on laboratory testing or clinicalsigns (e.g. persistent or recurrent high fever and other symptoms beyond2 or 3 days without signs of resolution); and

Severe dehydration, manifested as decreased physical or mental activity,dizziness, decreased urine output, and lethargy. (See WHO Guidelines forPharmacological Management of Pandemic Influenza A(H1N1) 2009 and otherInfluenza Viruses, Revised February 2010, Part I Recommendations, p. 5).

Uncomplicated influenza is a mild inflammation of the upper respiratorytract. Inflammation is part of the complex biological response of bodytissues to harmful stimuli damaging cells, such as viruses, toxins orirritants; it is a protective response that involves the immune system,blood vessels and numerous proteins. The purpose of inflammation is toeliminate the initial cause of cell injury, clear out dead and dyingcells and to initiate tissue repair. In uncomplicated influenza theinflammation is short lived and repair to the damaged epithelial celllining of the upper airways starts about 2-3 days after onset ofsymptoms.

In contrast to uncomplicated influenza, the inflammatory response insevere influenza is exaggerated or extended respectively. Rather thanbeing protective, the response becomes destructive. High levels ofpro-inflammatory proteins (cytokines) in the blood are an earlyindication of poor clinical outcomes in influenza patients (Lee, N. etal. 2011). In these patients the exaggerated inflammatory response canlead to damage of the blood vessels in the lung and other tissuesresulting in leakage of liquid into the tissue (oedema). Accumulation offluid and immune cells in the lungs can lead to pneumonia, acute lunginjury and ARDS and respiratory failure in severe cases. The exaggeratedinflammatory response in severe influenza can be viewed as a non-linearprocess where there is a critical point—a phase transition, or tippingpoint—when the normal inflammatory response becomes an abnormal or moredestructive response. In severe influenza, rather than going down aresolution path after peak symptoms at around day 3 after infection,patients progress to develop other respiratory complications, a processdriven by an exaggerated inflammatory response.

Armstrong S M et al., Endothelial activation and dysfunction in thepathogenesis of influenza A virus infection, Virulence, 2013; 4(6):537-542, review evidence in support of endothelial activation anddysfunction as a central feature preceding the development of severeinfluenza.

Using gene expression microarrays to compare the transcriptomic profilesof influenza-infected patients with severe, moderate and mild symptomswith febrile patients of unknown etiology, Hoang et al. (Patient-basedtranscriptome-wide analysis identify interferon and ubiquitinationpathways as potential predictors of influenza A disease severity, PloSOne 2014, 9: e111640e) reported that influenza-infected patients,regardless of their clinical outcomes, had a stronger induction ofantiviral and cytokine responses and a stronger attenuation of NK and Tcell responses in comparison with those with unknown etiology and thatinterferon and ubiquitination signalling were strongly attenuated inpatients with the most severe outcomes in comparison with those withmoderate and mild outcomes. This agrees with the inventors' own data,which show elevated cytokine levels in serum from patients hospitalisedfor severe influenza relative to influenza-infected individuals withoutsevere influenza (see Example 5 below).

Hoang et al., 2014 found p38 MAPK signalling to be up-regulated inmoderate as well as severe patients. MMP9, SOCS3, IFITMs, TLR10, RIG-I,CD244 and NCR3 were proposed as candidate genes for further studies.However, targeting individual cytokines is unlikely to have broadanti-inflammatory effects that are required, and redundancy in theinflammatory response system means that knocking out multiple cytokinessimultaneously is likely to be required for a therapy to be effective.

U.S. 2010/0151042 A1 (Liang et al.) claims a method for reducing theseverity, intensity or duration of complications or symptoms associatedwith an influenza infection, wherein the method comprises diagnosing asubject with the influenza infection; and concurrently administering tothe subject an effective amount of a cysteamine compound and a secondviral therapeutic. The complications associated with the influenza viralinfection may comprise rhinitis, bacterial infections, cardiaccomplications, neurologic complications, myositis, renal failure,pulmonary fibrosis, exacerbations of asthma, exacerbations of chronicobstructive pulmonary disease, empyema or heart failure. The secondviral therapeutic may be selected from the group consisting of:amantadine, rimantadine, ribavirin, idoxuridine, trifluridine,vidarabine, acyclovir, ganciclovir, foscarnet, zidovudine, didanosine,zalcitabine, stavudine, famciclovir, oseltamivir phosphate, zanamivir,valaciclovir, antitussives, mucolytics, expectorants, antipyretics,analgesics and nasal decongestants.

Immunomodulatory therapy for severe influenza has been reviewed by Huiet al., Adjunctive Therapies and Immunomodulatory Agents in theManagement of Severe Influenza, Antiviral Research, 2013; 98: 410-416;Hui & Lee, Adjunctive Therapies and Immunomodulatory Agents for SevereInfluenza, Influenza and Other Respiratory Viruses, 2013; 7 (suppl. 3):52-59; and Liu et al., Cellular & Molecular Immunology, 2015; 1-8, doi:10.1038/cmi.2015.74.

Darwish et al., Immunomodulatory Therapy for Severe Influenza, ExpertRev Anti Infect Ther., 2011 (July); 9(7): 807-22, doi:10.1586/eri.11.56, describe the influenza viral and host pathogenicitydeterminants, present the evidence supporting the use ofimmunomodulatory therapy to target the host inflammatory response as ameans to improve clinical outcome in severe influenza, and review theexperimental data on investigational immunomodulatory agents targetingthe host inflammatory response in severe influenza, including anti-TNFtherapy, statins, glucocorticoids, cyclooxygenase-2 inhibitors,macrolides, peroxisome proliferator-activated receptor agonists,AMP-activated protein kinase agonists and high mobility group box 1antagonists, concluding with a rationale for the use of mesenchymalstromal (stem) cells and angiopoietin-1 therapy against deleteriousinfluenza-induced host responses that mediate end-organ injury anddysfunction.

Fedson D S, Confronting an influenza pandemic with inexpensive genericagents: can it be done?, Lancet Infect. Dis., 2008; 8: 571-76, proposesresearch to determine whether statins, fibrates, chloroquines and othergeneric agents could mitigate the effects of an H5N1 avian influenza Apandemic. See also Fedson DS, Confronting the next influenza pandemicwith anti-inflammatory and immunomodulatory agents: why they are neededand how they might work, Influenza and Other Respiratory Viruses, 2009;3(4): 129-142, and Fedson DS,

Treating influenza with statins and other immunomodulatory agents,Antiviral Research, 2013; 99(3): 417-435.

An S C et al, Triple combinations of neuraminidase inhibitors, statinsand fibrates benefit the survival of patients with lethal avianinfluenza pandemic, Medical Hypotheses, 2011; 77(6): 1054-157,hypothesise that statins and fibrates, both of which haveanti-inflammatory and immunomodulatory effects and other multiplebiologic activities, may exhibit synergistic effects when they werecombined to neuraminidase inhibitors to treat the A (H5N1) virusinfections via inhibiting the production of either the earlyinflammatory mediators (e.g., many cytokine/chemokine) or the latemediator (e.g., High Mobility Group Box Protein 1), even showing theantiviral activities with the prevention of the development of antiviralresistance.

As described by Bermejo-Martin J F, et al, Macrolides for the treatmentof severe respiratory illness caused by novel H1N1 swine influenza viralstrains, J Infect Developing Countries, 2009; 3(3): 159-161, macrolidesare molecules with antibacterial activity that also have remarkablyanti-inflammatory properties. They exert both stimulatory and inhibitoryeffects on leukocytes. These effects seem to be related to theactivation state of the leukocytes, facilitating bacterial killing aswell as resolving local inflammation. The use of macrolides in thetreatment of severe disease caused by the novel H1N1 swine flureassortment influenza viruses is proposed, particularly in combinationwith antivirals, in order to diminish the systemic inflammatory responseleading to pneumonia and fatal outcome.

Sato K et al, Therapeutic Effect of Erythromycin on InfluenzaVirus-induced Lung Injury in Mice, Am J Respir Crit Care Med, 1998; 157:853-857, evaluated the use of erythromycin (EM), an antibiotic withpotent anti-inflammatory effects that is used for treating chronic lowerrespiratory tract infections, on influenza-virus-induced pneumonia inmice. It was found that the administration of EM at a dose of 3.3mg/kg/d (intraperitoneally, from Days 1-6 after infection) significantlyimproved the survival rate of mice infected with influenza virus, andthe survival rate of the virus-infected mice at Day 20 of infectionincreased in a dose-dependent fashion with EM administered to theanimals, from 14% among controls to 42% among animals given EM at 1.0mg/kg/d and 57% amongst those given EM at 3.3 mg/kg/d.

Börgeling et al., (Inhibition of p38 mitogen-activated protein kinaseimpairs influenza virus-induced primary and secondary host generesponses and protects mice from lethal H5N1 infection, J Biol Chem.2014 Jan 3;289(1):13-27), describe the use of SB 202190, a p38 MAPKinhibitor, to control interferon signalling in the very early stages ofinfluenza infection, and the resulting suppression of excessive cytokineexpression. SB 202190 is administered at the same time as exposure tovirus. The effects of viral infection concomitant with administration ofSB 202190 are analysed up to 2 days post-infection in influenza infectedmice. The data are limited to the effects of SB 202190 on cytokineexpression when administered at the onset of infection with influenzavirus (i.e. concomitant with exposure to virus). The effects of first SB202190 administration at time points following exposure to the virus arenot investigated and the effects of SB 202190 once the critical point(or tipping point) has been reached and the cytokine response becomesexaggerated and excessive are certainly not considered.

At the time of the present invention there remains an unmet need for atreatment to attenuate hypercytokinemia, and in particularhypercytokinemia associated with severe influenza virus infection.

SUMMARY OF THE INVENTION

As described below, especially with reference to the Examples, theinventors have discovered that p38 MAP kinase is upregulated in a numberof cellular signalling pathways that are very active in patients withsevere influenza in comparison with patients with mild or moderateinfluenza. In particular, it has been found that p38 MAP kinase isinvolved in a number of different non-metabolic signalling pathwayscomprising more than three nodes, in which 100% of the nodes areupregulated in patients with severe influenza versus patients with mildinfluenza, and at least 75% of the nodes are upregulated in patientswith severe influenza versus patients with H1N1 infection or moderateinfluenza.

The p38 MAP kinase is a node that is located high in a signallingcascade in a range of cell types (epithelial, endothelial and immune)which are all important in the pathology of severe influenza. Because ofthe rapid and progressive nature of severe influenza, targeting aprotein high in a signalling cascade is required to attenuateinflammatory mediator production quickly and reduce disease progression.

However, while numerous other targetable nodes in 95 pathway routes wereidentified by the inventors as potential targets for the treatment ofsevere influenza, it was unexpectedly found that p38 MAP kinaseinhibitors exhibit a dose-dependent knockdown effect on the release ofcytokines, particularly IP10, from endothelial cells treated with aviral conditioned medium that simulates the action of inflammatorymediators produced from influenza-infected epithelial cells, while otherpromising potential targets do not show the same effect. In particular,as disclosed by Example 4 below, while p38 MAP kinase inhibitors exhibitdose-dependent inhibition of IP10 in endothelial cells, as well asinhibitory effects on the release of inflammatory mediators from immunecells that are comparable with corticosteroids and macrolides, the nextmost promising target, namely mitogen-activated protein kinase (MEK), isnot effective in inhibiting IP10 release from endothelial cells andactually appears to increase levels of IP10 at higher drugconcentrations.

In addition, because severe influenza virus infection is oftencharacterised by hypercytokinemia, as explained herein, the Examples anddata presented below support the hypothesis that the p38 MAPK pathway isinvolved in hypercytokinemia more generally, i.e. hypercytokinemia thatis associated with conditions other than severe influenza virusinfection.

According to one aspect of the present invention therefore there isprovided a p38 MAPK inhibitor for use in the treatment or prevention ofhypercytokinemia. The hypercytokinemia may be associated with one ormore of the following: severe influenza virus infection;graft-versus-host disease (GVHD); acute respiratory distress syndrome(ARDS); sepsis; Ebola; smallpox; systemic inflammatory response syndrome(SIRS); bacterial infection; and cancer

According to another aspect of the present invention there is provided ap38 MAPK inhibitor for use in the treatment or prevention of severeinfluenza in a human patient.

According to another aspect of the present invention there is provided apharmaceutical composition comprising a p38 MAPK inhibitor for use inthe treatment or prevention of hypercytokinemia and/or for use in thetreatment or prevention of severe influenza in a human patient.

In a further aspect of the present invention there is provided a methodof treating or preventing hypercytokinemia and/or severe influenza in asubject (e.g. a human patient or an animal) in need thereof comprisingadministering to the subject (e.g. a human patient or an animal) apharmaceutically effective amount of a p38 MAPK inhibitor. Thehypercytokinemia may be associated with one or more of the following:severe influenza virus infection; graft-versus-host disease (GVHD);acute respiratory distress syndrome (ARDS); sepsis; Ebola; smallpox;systemic inflammatory response syndrome (SIRS); bacterial infection; andcancer

In particular, the p38 MAPK inhibitor has an anti-inflammatory and/or animmunomodulatory effect.

In particular, the p38 MAPK inhibitor may be used for the treatment orprevention of hypercytokinemia and/or severe influenza in a humanpatient by inhibiting the release of pro-inflammatory mediators fromendothelial cells and/or immune cells and/or epithelial cells (forexample endothelial cells and/or immune cells). Suitably, the p38 MAPkinase inhibitor may be administered in an amount effective forinhibiting the release of pro-inflammatory mediators from endothelialcells and/or immune cells and/or epithelial cells (for exampleendothelial cells and/or immune cells)

More specifically, the p38 MAPK inhibitor may be used for the treatmentor prevention of hypercytokinemia and/or severe influenza in a humanpatient by inhibiting the release of pro-inflammatory cytokines fromendothelial cells and/or immune cells and/or epithelial cells (forexample endothelial cells and/or immune cells). Suitably, the p38 MAPkinase inhibitor may be administered in an amount effective forinhibiting the release of pro-inflammatory cytokines from endothelialcells and/or immune cells and/or epithelial cells (for exampleendothelial cells and/or immune cells).

The p38 MAPK inhibitor may inhibit the release of one or more of, or allof: IL1-β, IL-6, IL-8, IL-10, IP10, TNFα, RANTES and/or MIP-1a (forexample one or more of, or all of: IL1-β, IL-6, IL-8, IL-10, IP10, TNFαand/or RANTES) from endothelial cells and/or immune cells. Inparticular, the p38 MAPK inhibitor may inhibit the release of IL-10.

Suitably, the p38 MAP kinase inhibitor may be administered in an amounteffective for inhibiting the release of one or more of, or all of:IL1-β, IL-6, IL-8, IL-10, IP10, TNFα, RANTES and/or MIP-1a fromendothelial cells and/or immune cells (for example one or more of, orall of: IL1-β, IL-6, IL-8, IL-10, IP10, TNFα and/or RANTES fromendothelial cells and/or immune cells). In particular, the p38 MAPkinase inhibitor may be administered in an amount effective forinhibiting the release of IL-10 from endothelial cells and/or immunecells.

In some embodiments, the p38 MAPK inhibitor may be used, in accordancewith the present invention, for the treatment or prevention ofhypercytokinemia and/or severe influenza in a human patient byinhibiting the release of IP10 from endothelial cells and/or immunecells.

Advantageously, the p38 MAPK inhibitor may exhibit dose-dependentinhibition of cytokine release from endothelial cells and/or immunecells.

As well as its inhibitory effects on cytokine release from endothelialcells, the p38 MAPK inhibitor may also act to inhibit the release ofpro-inflammatory cytokines from immune cells, which are typicallylocated close to endothelial cells in the lower respiratory tract.Suitably, the p38 MAP kinase inhibitor may be administered in an amounteffective for inhibiting cytokine release from endothelial cells. Inparticular, the p38 MAP kinase inhibitor may be administered in anamount effective for inhibiting cytokine release from endothelial cellsand inhibiting pro-inflammatory cytokines release from immune cells.

Suitably, the p38 MAPK inhibitor is of Formula I below or apharmaceutically acceptable salt or solvate thereof:

wherein R is C₁₋₃alkyl, which is optionally substituted by one or moreof halo, NR¹R² or hydroxy, and R¹ and R² are independently H, halo orC₁₋₃alkyl, which is optionally substituted by one or more F.

For example, R may be optionally substituted by one, two or three ofhalo, NR¹R² or hydroxyl; or may be optionally substituted by one or twoof halo, NR¹R² or hydroxyl; or may be optionally substituted by one ofhalo, NR¹R² or hydroxyl. When R is optionally substituted by more thanone (for example two or three) halo, NR¹R² and/or hydroxyl, thosesubstituents may be independently selected from that list.

When R¹ and/or R² is a C₁₋₃alkyl optionally substituted by one or moreF, each C₁₋₃alkyl may, for example, be optionally substituted by one,two, or three F; be optionally substituted by one or two F; beoptionally substituted by one or three F; be optionally substituted byone F; or be optionally substituted by three F.

In some embodiments, R is methyl or ethyl. In another embodiment, R maybe propyl.

R may be substituted by one or more fluoride atoms.

In some embodiments, R may be substituted by hydroxy. For example, R maybe ω-substituted alkyl, i.e. ω-hydroxyalkyl. Thus, in one embodiment, Rmay be 3-propanol.

Suitably R¹ and R² may be independently selected from H and CH₃.

In some embodiments, R may be C₁₋₃alkyl, which is substituted by one ormore of halo, NR¹R² or hydroxy, and R¹ and R² are independently H, haloor C₁₋₃alkyl, which is optionally substituted by one or more F. Forexample, R may be C₁₋₃alkyl, which is substituted by one, two or threeof halo, NR¹R² or hydroxy, and R¹ and R² are independently H, halo orC₁₋₃alkyl, which is optionally substituted by one, two or three F.

In some embodiments, the p38 MAPK inhibitor may be of Formula II or apharmaceutically acceptable salt or solvate thereof:

Alternatively, the p38 MAPK inhibitor may be of Formula III below or apharmaceutically acceptable salt or solvate thereof:

The compounds of Formula II and III of the present invention, and thesynthesis of those compounds, is disclosed in WO 2004/076450 A1 (seeExamples 18 and 8, respectively). The contents of WO 2004/076450 A1 arehereby incorporated by reference.

In some embodiments, the p38 MAPK inhibitor may be of Formula I, or apharmaceutically acceptable salt or solvate thereof, as defined above,with the proviso the compound is not a compound of Formula III, i.e. nota compound having the following structure:

By “severe influenza” herein is meant an illness caused by any influenzavirus and leading to lower respiratory tract clinical disease and/orlower respiratory tract inflammation and/or hypercytokinemia (e.g. lungor systemic). As described above, severe influenza is distinct fromuncomplicated (mild or moderate) influenza in which the patienttypically has clinically tolerable upper and lower respiratory tractsymptoms such, for example, as nasal congestion, sneezing, rhinorrhoea,pyrexia (fever) and cough and sputum production, and from which apatient normally recovers naturally without the need for therapeuticintervention. In severe influenza, the patient's inflammatory responseto the viral infection is grossly exaggerated or extended, and thepatient develops more severe respiratory tract symptoms or clinicalcomplications that may require hospitalization and sometimes result indeath. In such cases, early therapeutic intervention is criticallyimportant. As indicated by the WHO definition of severe influenza, awide range of complications can be caused by influenza virus infectionof the upper respiratory tract (nasal passages, sinuses, throat) andlower respiratory tract (lungs). Different patients with severeinfluenza may therefore present with a wide range of different symptomsor signs of the disease. The various symptoms or signs discussed belowthat are characteristic of severe influenza may be observable ordetectable after 2 days of illness; typically, within 2-9 days ofillness.

For instance, the severe influenza may be characterised byhypercytokinemia. Typically, the hypercytokinemia may involve elevatedlevels of one or more cytokines.

In some embodiments, the cytokines may comprise one or more of IL-8,IL-7, IL-6, Eotaxin, IP10, MCP1, MCP4, VEGF and MIP-la (for example oneor more of IL-8, IL-7, IL-6, Eotaxin, IP10, MCP1, MCP4 and VEGF). Inparticular, in some embodiments, the cytokines may comprise one or moreof IL-8, Eotaxin, IP10, IL-7 and MIP-1a (for example one or more ofIL-8, Eotaxin, IP10 and IL-7).

In some embodiments, the cytokines may comprise one or more of IL-6,IL-8 and IP10 (see Lee N. et al., 2011; Lee N. et al., Viral clearanceand inflammatory response patterns in adults hospitalised for pandemic2009 influenza A (H1N1) virus pneumonia, Antiviral Therapy, 2011; 16:237-47).

In some embodiments, the hypercytokinemia may involve an elevated levelof IL-6 of greater than about 1.5 or 2 times its plasma reference range(<3.1 pg/ml)—in some embodiments greater than about 10, 15 or 30 times,up to about 54 times or more. Thus, the hypercytokinemia may involve anelevated level of IL-6 of greater than about 4.7 pg/ml. Moreparticularly, the hypercytokinemia involve an elevated level of IL-6 ofgreater than about 4.7 pg/ml in the case of seasonal flu, and greaterthan about 7.8 pg/ml in the case of pandemic flu.

In some embodiments, the hypercytokinemia may involve an elevated levelof IL-8 of greater than about 1 or 2 times its plasma reference range(<5.0 pg/ml)—in some embodiments greater than about 4 times, up to about8, 10 or 12 times or more. Thus, the hypercytokinemia may involve anelevated level of IL-8 greater than about 5.0 pg/ml. More particularly,the hypercytokinemia may involve an elevated level of IL-8 of greaterthan about 5.0 pg/ml in the case of seasonal flu, and greater than about11.6 pg/mL in the case of pandemic flu.

In some embodiments, the hypercytokinemia may involve an elevated levelof IP-10 of greater than about 1 or 2 times its plasma reference range(202-1480 pg/ml)—in some embodiments greater than 1.1 times, greaterthan 1.5 times or greater than about 2, 3, 4, 5, 6, 7 or 8 times, up toabout 10, 20 or 30 times or more. Thus, the hypercytokinemia may involvean elevated level of IP-10 of greater than about 835 pg/ml. Moreparticularly, the hypercytokinemia may involve an elevated level ofIP-10 of greater than about 835 pg/ml in the case of pandemic flu, andgreater than about 1476 pg/ml in the case of seasonal flu.

In some embodiments, the hypercytokinemia may involve an elevated levelof MCP-1 of greater than about 1 or 2 times its plasma reference range(<10.0-57.0 pg/ml)—in some embodiments greater than about 4 times, up toabout 5.5 times or more. Thus, the hypercytokinemia may involve anelevated level of MCP-1 of greater than about 52.9 pg/ml. Moreparticularly, the hypercytokinemia may involve an elevated level ofMCP-1 of greater than about 52.9 pg/ml in the case of pandemic flu, andgreater than about 64.8 pg/ml in the case of seasonal flu.

In some embodiments, the hypercytokinemia may involve an elevated levelof sTNFR-1 of greater than about 1 or 2 times its plasma reference range(484-1407 pg/ml), up to about 2.5 times or more. Thus, thehypercytokinemia may involve an elevated level of sTNFR-1 of greaterthan about 1099.4 pg/ml. More particularly, the hypercytokinemia mayinvolve an elevated level of sTNFR-1 of greater than about 1099.4 pg/mlin the case of seasonal flu, and greater than about 1250.7 pg/ml in thecase of pandemic flu.

In some embodiments, the hypercytokinemia may involve an elevated levelof MIG of greater than about 1 or than 2 times its plasma referencerange (48.0-482.0 pg/ml)—in some embodiments greater than 1.1 times,greater than 1.5 times or greater than 2 times, up to about 15, 40 or 50times or more. Thus, the hypercytokinemia may involve an elevated levelof MIG of greater than about 103.8 pg/ml. More particularly, thehypercytokinemia may involve an elevated level of MIG of greater thanabout 103.8 pg/ml in the case of seasonal flu, and greater than about118.7 pg/ml in the case of pandemic flu.

In some embodiments, the hypercytokinemia may involve an elevated levelof IL-17A of greater than about 1, 1.5 or 2 times its plasma referencerange (<10.0 pg/ml)—in some embodiments greater than 4 times, greaterthan 5 times or greater than 6 times, up to about 7 times or more. Thus,the hypercytokinemia may involve an elevated level of IL-17A of greaterthan about 5.0 pg/ml. More particularly, the hypercytokinemia mayinvolve an elevated level of IL-17A of greater than about 5.0 pg/ml inthe case of pandemic flu and greater than about 9.3 pg/ml in the case ofseasonal flu.

The severe influenza may be characterised in some embodiments bysustained activation of the pro-inflammatory cytokines (IL-6, IL-8, IP10, MCP-1, sTNFR-1 and/or MIP-la; for example IL-6, IL-8, IP10, MCP-1and/or sTNFR-1). The present invention provides a p38 MAPK inhibitor foruse as disclosed herein (for example for use in the treatment orprevention of hypercytokinemia (e.g. hypercytokinemia associated withsevere influenza virus infection) and/or for use in the treatment orprevention of severe influenza), wherein the p38 MAPK inhibitor inhibitsthe release of pro-inflammatory cytokines from endothelial cells and/orimmune cells and/or epithelial cells; for example from endothelial cellsand immune cells; or from endothelial cells, immune cells and epithelialcells. The p38 MAPK inhibitor may be administered in combination with afurther agent. In particular, the present invention also provides a p38MAPK inhibitor administered in combination with an antimicrobial agent(such as an antiviral agent), or an anticancer agent, and preferablywith an antimicrobial agent (such as an antiviral agent) ,wherein thep38 MAPK inhibitor inhibits the release of pro-inflammatory cytokinesfrom endothelial cells and/or immune cells and/or epithelial cells; forexample from endothelial cells and immune cells; or from endothelialcells, immune cells and epithelial cells..

The present invention also provides a method of treating or preventinghypercytokinemia and/or severe influenza in a subject (e.g. a humanpatient or an animal) in need thereof as described herein (for example amethod of treating or preventing hypercytokinemia (e.g.

hypercytokinemia associated with severe influenza virus infection)and/or a method of treating or preventing severe influenza) wherein thep38 MAPK inhibitor inhibits the release of pro-inflammatory cytokinesfrom endothelial cells and/or immune cells and/or epithelial cells; forexample from endothelial cells and immune cells; or from endothelialcells, immune cells and epithelial cells. The p38 MAPK inhibitor may beadministered in combination with a further agent. In particular, thepresent invention also provides a method of treating or preventinghypercytokinemia and/or severe influenza in a subject (e.g. a humanpatient or an animal) in need thereof as described herein, wherein thep38 MAPK inhibitor is administered in combination with an antimicrobialagent (such as an antiviral agent, or an anticancer agent, andpreferably with an antimicrobial agent, such as an antiviral agent), andwherein the p38 MAPK inhibitor inhibits the release of pro-inflammatorycytokines from endothelial cells and/or immune cells and/or epithelialcells; for example from endothelial cells and immune cells; or fromendothelial cells, immune cells and epithelial cells.

Levels of cytokines may be detected in the patient's whole blood, serum,plasma, nasal lavage, nasal secretions or bronchiolar alveolar lavage.Levels of cytokines may be quantified using any suitable technique knownto those skilled in the art. Suitably, an enzyme-linked immunosorbentassay (ELISA) or fluorescent automated cell sorting (FACs) may be used.By way of example, a chemiluminescent immunoassay system such, forexample, as that which is available from Meso Scale Diagnostics LLC(http://web.archive.org/web/20160522190937/https://www.mesoscale.com/)may be employed for its speed and sensitivity.

Further or alternatively, the severe influenza may be accompanied bysignificantly higher total white blood cell counts. A patient withsevere influenza may have significantly higher absolute neutrophilcounts than a patient with mild or moderate influenza. Typically, apatient with severe influenza after 2-9 days of illness may have aneutrophil count in the range 2.1-24.5×10³/μl (as compared with apatient with moderate influenza after 1-9 days of illness who may have aneutrophil count in the range 0.62-10.88×10³/μl or a patient with mildinfluenza after 3-8 days of illness who may have a neutrophil count inthe range 0.5-6.5×10³/μl). In some embodiments, the absolute plateletcount may be significantly lower in patients with severe disease after2-9 days of illness, e.g. 27-250×10³/μl (as compared with a patient withmoderate influenza after 1-9 days of illness who may have a plateletcount in the range 55-345×10³/μl or a patient with mild influenza after3-8 days of illness who may have a platelet count in the range79-370×10³/μl) (Hoang et al, 2014).

Further, or alternatively, the severe influenza may be characterised bysymptoms or signs of hypoxemia or cardiopulmonary insufficiency. In someembodiments, the patient may have an arterial oxygen saturation of ≤92%on room air by a transcutaneous method. Typically, the symptoms or signsof hypoxemia or cardiopulmonary insufficiency may include one or more ofdyspnoea, tachypnoea, cyanosis, low blood pressure (designated as belownormal range for age and sex) and tachycardia.

In some embodiments, the patient may have tachypnoea (respiratoryrate≥30 for ages≥12 years, rate≥40 for ages 6 to 12 years, rate≥45 forages 3 to 6 years, rate≥50 for ages 1 to 3 years).

In some embodiments, the patient may have or show signs of discomfortwith breathing or dyspnoea (unable to speak full sentences, appearbreathless, using accessory respiratory muscles).

Further, or alternatively, the severe influenza may be characterised bycomorbidity with a lower respiratory disorder with or withoutradiological pulmonary infiltrates.

Further, or alternatively, the severe influenza may be characterised bysymptoms or signs suggesting CNS and/or peripheral neuromusculardisorders such as, for example, encephalitis, myelitis orrhabdomyolysis, including altered mental state, unconsciousness,drowsiness, difficult to awaken, recurring convulsions, confusion,muscle pain, severe weakness, paralysis and sensory abnormalities (e.g.tingling in limbs, loss of normal pain sensation).

Still further, or alternatively, the severe influenza may becharacterised by severe dehydration. The WHO defines severe dehydrationin adults as >9% body weight loss (in children>15%) (K. Sinha and M.Davenport (eds.), Handbook of Pediatric Surgery, doi:10.1007/978-1-84882-132-3_2.1, Springer-Verlag London Limited 2010).According to the present invention, the severe influenza mayinvolve >9%, for example 10-15%, loss of body fluids.

Further, or alternatively, the severe influenza may be characterised byabnormal levels of fatigue and/or lethargy.

Further, or alternatively, the severe influenza may be characterised bythe presence of radiological pulmonary infiltrate.

Still further, or alternatively, the severe influenza involves evidenceof sustained viral infection or replication. In some embodiments, thepatient may exhibit more than 2 days of constant or increasing viralreplication that can be assayed using standard laboratory methods ordiagnosed by the identification of persistent or worsening symptoms. Insome embodiments, the patient may exhibit 3, 4, 5 or more days ofconstant or increasing viral replication. Thus, in some embodiments, thesevere influenza in accordance with the present invention may becharacterized by symptoms that persist or recur for more than 2, 3, 4, 5or more days without signs of resolution. The symptoms that persist orrecur may include fever (i.e. a temperature greater than 100° F./38°C.), lethargy, achiness, congestion, cough, sinus congestion, sinusdrainage or upper respiratory congestion or inflammation.

Further, or alternatively, the severe influenza may be characterised bya secondary bacterial infection.

Further, or alternatively, the severe influenza may be characterised bya lower respiratory tract disorder or inflammation.

Further, or alternatively, the severe influenza may be characterised bymono- or multi-organ failure (e.g. respiratory failure or renal failure)or septic shock.

In some embodiments, the patient may be an infant (i.e. less than oneyear old) or elderly (i.e. 65 years old or more) or may be a pregnantwoman.

In some embodiments, the human patient may have one or more underlyingcomorbidities that predispose the patient to severe influenza. Forexample, the patient may be immunocompromised, or may suffer from COPD,severe genetic anaemia, asthma or diabetes, chronic hepatic or renalinsufficiency, obesity or a cardiovascular disorder or condition.

Thus, in some embodiments, the p38 MAPK inhibitor may be administered tothe patient prophylactically, especially where the patient is in a“high-risk” or “at-risk” group as mentioned in the preceding paragraphs.

The p38 MAPK inhibitor may be administered to the patient afterhypercytokinemia has developed, for example after the patient has beenadmitted to hospital. The p38 MAPK inhibitor may be administered afterthe critical point (threshold or tipping point) where the normalinflammatory response becomes an abnormal response. The p38 MAPKinhibitor may be administered to prevent hypercytokinemia developing,i.e. before the critical point is reached. This critical point ischaracterised by a threshold level of cytokines. The threshold level ofcytokines will be, in part, dependent on the patient. The p38 MAPKinhibitor may be first administered at least 8 hours after an immuneresponse is triggered e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, or 47 hours after an immune response istriggered: The p38 MAPK inhibitor may be first administered to thepatient at least 48 hours after an immune response is triggered, atleast 60 hours, at least 72 hours, at least 84 hours, at least 96 hours,at least 108 hours, at least 120 hours, at least 132 hours, at least 144hours, at least 156 hours, at least 168 hours, at least 180 hours, atleast 192 hours, at least 204 hours at least 216 hours, at least 228hours, at least 240 hours after an immune response is triggered. The p38MAPK inhibitor may be administered on multiple occasions at multipletime points after an immune response is triggered. The immune responsemay be triggered for example by exposure to a pathogen, for exampleinfluenza virus infection leading to severe influenza virus infection,or the immune response may be triggered by cancer, or may be triggeredby an autoimmune response.

The p38 MAPK inhibitor may be administered at a dose between about 10mgand about 1000 mg, for example between 10 mg and 1000 mg or between 10mg and 400 mg. Up to 1000 mg may be administered per day in single ormultiple doses for between 1 and 10 days, e.g. for 2 days, 3 days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days.

The present invention also provides a p38 MAPK inhibitor administered incombination with a further agent. In particular, the present inventionalso provides a p38 MAPK inhibitor administered in combination with anantimicrobial agent, such as an antiviral agent, or an anticancer agent,and preferably with an antimicrobial agent, such as an antiviral agent.Where the p38 MAPK inhibitor is administered in combination with afurther agent (for example an antimicrobial agent, such as an antiviralagent, or an anticancer agent), the p38 MAPK inhibitor and the furtheragent may be administered simultaneously, or the p38 MAPK inhibitor andthe further agent may be administered sequentially, or the p38 MAPKinhibitor and the further agent may be administered separately. Inparticular, the further agent may be administered prior to the p38 MAPKinhibitor.

Preferably, the p38 MAPK inhibitor and the further agent (for example anantimicrobial agent, such as an antiviral agent, or an anticancer agent)may be administered simultaneously, for example in a single dosage formfor simultaneous administration, e.g. for simultaneous oraladministration. A single dosage form may also be referred to as a ‘unitdose’ or ‘unit dosage form’. A single dosage form comprising a p38 MAPKinhibitor and a further agent comprises a mixture of the p38 MAPKinhibitor and the further agent, and optionally also inactive componentssuch as pharmaceutically acceptable excipients. The single dosage formfor simultaneous oral administration may, for example, be a tablet,powder or capsule.

The present invention also provides a p38 MAPK inhibitor for use asdescribed herein (for example for use in the treatment or prevention ofhypercytokinemia (e.g. hypercytokinemia associated with severe influenzavirus infection) and/or for use in the treatment or prevention of severeinfluenza), wherein the p38 MAPK inhibitor is administered systemicallyor non-systemically, and preferably systemically, and in particularorally.

The present invention also provides a pharmaceutical composition for useas described herein (for example for use in the treatment or preventionof hypercytokinemia (e.g. hypercytokinemia associated with severeinfluenza virus infection) and/or for use in the treatment or preventionof severe influenza), wherein the pharmaceutical composition isformulated for oral administration.

Embodiments of the invention providing a p38 MAPK inhibitor (for examplea p38 MAPK inhibitor of formula I) administered in combination with anantiviral agent (for example a neuraminidase inhibitor antiviral agents(e.g. oseltamivir phosphate)) are especially preferred as embodiments ofthe invention because the inventors have surprisingly found that a p38MAPK inhibitor does not interfere with the effect of an antiviral agent(for example a neuraminidase inhibitor antiviral agents (e.g.oseltamivir phosphate)), and an antiviral agent (for example aneuraminidase inhibitor antiviral agents (e.g. oseltamivir phosphate))does not interfere with the effect of a p38 MAPK inhibitor.

In another aspect of the present invention there is provided a method oftreatment as described herein (for example for a method of treatment orprevention of hypercytokinemia (e.g. hypercytokinemia associated withsevere influenza virus infection) and/or a method of treatment orprevention of severe influenza) in a subject (e.g. a human patient oranimal) which comprises administering a therapeutically effective amountof a p38 MAP kinase inhibitor to a subject in need thereof wherein thep38 MAPK inhibitor is administered systemically or non-systemically tothe subject (e.g. a human patient or animal), and preferablysystemically, and in particular orally.

DETAILED DESCRIPTION OF THE INVENTION

p38 MAPK inhibitors are an established class of active agents (see e.g.Zarubin and Han, Cell Research (2005) 15, 11-18.doi:10.1038/sj.cr.7290257). A wide range of p38 MAPK inhibitors areavailable to those skilled in the art (see e.g. Lee, et al., Inhibitionof p38 MAP kinase as a therapeutic strategy, Immunopharmacol., 2000;47(2-3): 185-201. doi:10.1016/S0162-3109(00)00206-X, which isincorporated herein by reference). Examples of p38 MAPK inhibitorsinclude VX-745, VX-702, RO-4402257, SCIO-469, BIRB-746, SD-0006,PH-787804, AMG-548, SB-681323 (Dilmapimod), LY2228820, GW-856553,RWJ67657 and BCT-197 (Xing, L., MAP Kinase 2015, Vol. 4:5508, which isincorporated herein by reference). These are examples of p38 MAPKinhibitors that have reached human trials.

A preferred p38 MAPK inhibitor of the present invention is representedby Formula I, or a pharmaceutically salt or solvate thereof:

wherein R is as defined above.

The p38 MAPK inhibitor of Formula I may have Formula II, or apharmaceutically acceptable salt or solvate thereof:

The p38 MAPK inhibitor of Formula I may have Formula III or apharmaceutically acceptable salt or solvate thereof:

The p38 MAPK inhibitors of Formulae I, II and III have a similarstructure and are all thought to interact with the P38 MAPK active siteto inhibit p38 MAPK mediated signalling. The inhibitors of Formulae I,II and III are thought specifically to inhibit p38α and p38β.

The compounds of Formula I may have particular advantages as p38 MAPKinhibitors, such as those described below. Certain compounds within thescope of Formula I may have particularly favourable pharmacokineticproperties (and in particular favourable pharmacokinetic propertiesmaking them especially suitable for oral administration), and/orfavourable properties due to having a low side-effect profile and/or lowtoxicity. Such compounds include compounds of Formula I in which R is asubstituted C₁₋₃alkyl group and/or in which R is a substituted orunsubstituted propyl moiety, for example the compound of Formula II inwhich R is propan-3-ol.

The use of a p38 MAPK inhibitor in accordance with the present inventionaims to treat or prevent hypercytokinemia. As set out abovehypercytokinemia is characterised by an exaggerated inflammatoryresponse and there is a critical point (i.e. threshold or tipping point)where the normal response becomes an abnormal response andhypercytokinemia develops. This critical point is characterised by athreshold level of cytokines. The threshold level of cytokines will be,in part, dependent on the patient.

Hypercytokinemia may be associated with a number of infectiousconditions and non-infectious conditions. In particular,hypercytokinemia may be associated with severe influenza virusinfection; graft-versus-host disease (GVHD); acute respiratory distresssyndrome (ARDS); sepsis; Ebola; smallpox; systemic inflammatory responsesyndrome (SIRS); bacterial infection; and cancer.

In particular, the use of a p38 MAPK inhibitor in accordance with thepresent invention aims to attenuate the inflammatory response in apatient with severe influenza, rather than ablate it, with the objectiveof blunting the damaging effects of the out-of-control inflammation,whilst preserving its protective and disease pro-resolution effects.Total ablation of inflammation would be likely to promote mortality insevere influenza, whereas attenuation of this explosive process shouldprovide protection against the damaging effects caused by excessinflammatory responses whilst preserving the host's essential innatedefence activities. The present invention therefore aims to “re-balancethe system” rather than knock out components in their entirety.

p38 MAPK inhibitors that are especially useful at “re-balancing thesystem” by attenuating the inflammatory response in a patient withhypercytokinemia associated with severe influenza include the compoundsof Formula I, and in particular the compounds of Formula I in which R issubstituted C₁₋₃alkyl group and/or in which R is a substituted orunsubstituted propyl group, for example the compound of Formula II inwhich R is propan-3-ol. Such compounds may be very effective atattenuating the inflammatory response in a patient with severe influenzabut do not ablate the immune response in its entirety; for example asshown by the results of Examples 6 and 8 in FIGS. 17, 18, 20 and 21,below.

Furthermore, from Example 6 and Example 8 below, the compound of FormulaII exhibited consistently better effects than the compound of FormulaIII at attenuating the inflammatory response in endothelial and immunecells when simple (TNFa plus IL-6) and complex (HBEC or A459 viral soup)stimuli were applied to simulate the interaction of inflammatorymediators produced by influenza-infected epithelial cells on endothelialcells and immune cell. Thus, in certain embodiments, the compound ofFormula I is preferably one in which R is a substituted C₁₋₃alkyl groupand/or in which R is a substituted or unsubstituted propyl moiety, andmore preferably the compound of Formula I is the compound of Formula II.

The p38 MAPK inhibitor may be administered in combination with anantimicrobial agent. The antimicrobial agent may be any one, or more, ofthe following: an antibacterial, an antibiotic, an antifungal, anantiviral, an antiparasitic, and/or an antimicrobial monoclonalantibody. An antimicrobial monoclonal antibody is any monoclonalantibody that is used to attempt to treat or prevent microbial disease.

In particular, the p38 MAPK inhibitor may be administered in combinationwith an antiviral agent. The antiviral agent may be any one, or more, ofthe following or pharmaceutically acceptable salts thereof: amantadine;rimantadine; ribavirin; idoxuridine; trifluridine; vidarabine;acyclovir; ganciclovir; foscarnet; zidovudine; didanosine; zalcitabine;stavudine;

famciclovir; valaciclovir; antitussives; mucolytics; expectorants;antipyretics; analgesics and/or nasal decongestants. Preferably theantiviral agent is a neuraminidase inhibitor such as: oseltamivirphosphate, zanamivir, peramivir and/or laninamivir. In a preferredaspect the p38 MAPK inhibitor is administered in combination withoseltamivir or a pharmaceutically acceptable salt thereof (e.g.oseltamivir phosphate). While not wishing to be bound by theory it ishypothesised that the oseltamivir phosphate combats the influenzainfection (has an antiviral effect) while the p38 MAPK inhibitor has animmunomodulatory effect against hypercytokinemia.

It has been surprisingly found by the present inventors that p38 MAPKinhibitors and antiviral agents (such as neuraminidase inhibitorantiviral agents (e.g. oseltamivir phosphate)), and more especially p38MAPK inhibitors of Formula I and neuraminidase inhibitor antiviralagents (e.g. oseltamivir phosphate), do not significantly interfere witheach other's activity, i.e. antiviral agents do not negate theattenuating anti-inflammatory effects of p38 MAPK inhibitors; and thep38 MAPK inhibitors do not negate the antiviral effects of anti-viralagents. For example, the results of Example 9 in FIGS. 22 and 23 belowshow that when oseltamivir was combined with compounds of Formula II,TCID₅₀ was reduced to the level seen with oseltamivir on its own,indicating that p38MAPK inhibitor therapy may be used in combinationwith oseltamivir without materially impacting its antiviral activity(FIG. 22); and also that oseltamivir had no observed effect on theanti-inflammatory properties of the compound of Formula II (FIG. 23).

The non-interference between these agents is especially advantageousbecause, as described above, the anti-inflammatory properties of the p38MAPK inhibitor compound may be used in accordance with the presentinvention to “re-balance the system” of inflammation in the body whenused in the treatment of hypercytokinemia associated with severeinfluenza. Thus any increase or decrease in the anti-inflammatoryproperties of the p38 MAPK inhibitor could lead to a loss of thebalancing effect of the p38 MAPK inhibitor compound, e.g. a decrease inthe anti-inflammatory properties of the p38 MAPK inhibitor may notsufficiently blunt the damaging effects of the out-of-controlinflammation, and an increase in the anti-inflammatory properties of thep38 MAPK inhibitor may ablate the necessary protective and diseasepro-resolution effects of the body's inflammatory response. It is alsoadvantageous that the p38 MAPK inhibitor compound does not adverselyaffect the anti-viral effects of the antiviral agent.

As such, in another embodiment, the present invention provides a p38MAPK inhibitor for use in the present invention (for example for use inthe treatment or prevention of hypercytokinemia (e.g. hypercytokinemiaassociated with severe influenza virus infection) and/or for use in thetreatment or prevention of severe influenza), wherein the p38 MAPKinhibitor is administered to a patient who has been administered anantiviral agent for the treatment of severe flu. In such embodiments,the p38 MAPK inhibitor may be administered, for example, within 72hours, 60 hours, 48 hours, 36 hours, 24 hours, 16 hours, 12 hours, 10hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour of administrationof the antiviral agent. The antiviral agent may be one as describedabove (e.g. oseltamivir phosphate).

In a further embodiment, the present invention provides a p38 MAPKinhibitor for use in the present invention (for example for use in thetreatment or prevention of hypercytokinemia (e.g. hypercytokinemiaassociated with severe influenza virus infection) and/or for use in thetreatment or prevention of severe influenza), wherein the p38 MAPKinhibitor is administered to a patient who is undergoing treatment ofsevere flu comprising administering an antiviral agent. In suchembodiments, the patient may have undergone treatment of severe flucomprising administering an antiviral agent for at least 1 hour (forexample 1 hours, 2 hours, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 9 days or 10 days), at least 2hours (for example 2 hours, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 9 days or 10 days), at least 4hours (for example 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4days, 5 days, 6 days, 7 days, 9 days or 10 days), at least 8 hours (forexample 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 9 days or 10 days), at least 12 hours (for example 12hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 9 days or10 days), at least 1 day (1 day, 2 days, 3 days, 4 days, 5 days, 6 days,7 days, 9 days or 10 days), at least 2 days (for example 2 days, 3 days,4 days, 5 days, 6 days, 7 days, 9 days or 10 days), or 3 days (forexample 3 days, 4 days, 5 days, 6 days, 7 days, 9 days or 10 days),before treatment with the p38 MAPK inhibitor. The antiviral agent may beone as described above (e.g. oseltamivir phosphate).

In another embodiment, the present invention provides a method oftreating or preventing hypercytokinemia in a human patient as describedherein (for example a method of treating or preventing hypercytokinemia(e.g. hypercytokinemia associated with severe influenza virus infection)and/or a method of treating or preventing severe influenza), wherein thep38 MAPK inhibitor is administered to a patient who has beenadministered an antiviral agent for the treatment of severe flu. In suchembodiments, the p38 MAPK inhibitor may be administered, for example,within 72 hours, 60 hours, 48 hours, 36 hours, 24 hours, 16 hours, 12hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour ofadministration of the antiviral agent. The antiviral agent may be one asdescribed above (e.g. oseltamivir phosphate).

In embodiments where the antiviral agent is oseltamivir or a salttherefore, and in particular in embodiments where the antiviral agent isoseltamivir phosphate, the oseltamivir or a salt thereof (e.g. theoseltamivir phosphate) may be administered at a dose from about 1 mg andto 200 mg, for example between 5 mg and 100 mg (e.g. 5, 6, 7, 8, 9, 10,12, 15, 20, 30, 45, 50, 75 or 100 mg), and preferably between 6 mg and75 mg preferably 6, 12, 30, 45 or 75 mg). Single or multiple doses ofthe antiviral agent may be administered in a day, for example 1 dose perday, or 2 doses per day.

In embodiments where the antiviral agent is oseltamivir or a salttherefore, and in particular in embodiments where the antiviral agent isoseltamivir phosphate, the oseltamivir or a salt thereof (e.g. theoseltamivir phosphate) may be administered as a dose to achieve a bloodplasma level of oseltamivir carboxylate of 1 to 750 μg/L, preferably 5to 600 μg/L, preferably 10 to 500 μg/L, more preferably 25 to 500 μg/L,and more preferably 50 to 500 μg/L, for example 100 to 400 μg/L. Singleor multiple doses of the antiviral agent may be administered in a day,for example 1 dose per day, or two doses per day, to achieve a bloodplasma level of oseltamivir carboxylate as described above.

In a further embodiment, the present invention provides a method oftreating or preventing hypercytokinemia in a human patient as describedherein (for example a method of treating or preventing hypercytokinemia(e.g. hypercytokinemia associated with severe influenza virus infection)and/or a method of treating or preventing severe influenza), wherein thep38 MAPK inhibitor is administered to a patient who is undergoingtreatment of severe flu comprising administering an antiviral agent. Insuch embodiments, the patient may have undergone treatment of severe flucomprising administering an antiviral agent for at least 1 hour (forexample 1 hours, 2 hours, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 9 days or 10 days), at least 2hours (for example 2 hours, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 9 days or 10 days), at least 4hours (for example 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4days, 5 days, 6 days, 7 days, 9 days or 10 days), at least 8 hours (forexample 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 9 days or 10 days), at least 12 hours (for example 12hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 9 days or10 days), at least 1 day (1 day, 2 days, 3 days, 4 days, 5 days, 6 days,7 days, 9 days or 10 days), at least 2 days (for example 2 days, 3 days,4 days, 5 days, 6 days, 7 days, 9 days or 10 days), or 3 days (forexample 3 days, 4 days, 5 days, 6 days, 7 days, 9 days or 10 days),before treatment with the p38 MAPK inhibitor. The antiviral agent may beone as described above (e.g. oseltamivir phosphate).

The p38 MAPK inhibitor may be administered in combination with ananticancer agent. An anticancer agent is any agent that is used toattempt to treat or prevent cancer.

The p38 MAPK inhibitor may be administered systemically ornon-systemically, such as orally, or topically, including epidermally,bucally, intranasally or via inhalation (aerosol), or both intranasallyand via inhalation or parenterally (e.g. intravenously, subcutaneously)or in combination topically and parenterally.

As used herein “topically” includes non-systemic administration. Thisincludes the application of a compound externally to the epidermis orthe buccal cavity and/or the instillation of such a compound into theear, eye and nose.

As used herein “systemic administration” refers to oral, intravenous,intraperitoneal and intramuscular administration, subcutaneousintranasal, intra-rectal or intravaginal.

In particular the p38 MAPK inhibitor may be administered intravenously.Preferably the p38 MAPK inhibitor is administered orally. Oraladministration allows for a systemic rather than localized effect. Thisis advantageous as it may allow for all cell types associated withand/or affected by the hypercytokemia (in particular hypercytokemiaassociated with severe flu) to be treated with the p38 MAPK inhibitor(i.e. endothelial cells, epithelial cells and/or immune cells). Inaddition, oral formulations, for example tablets, are easy to take andso are associated with improved patient compliance. Oral administrationis particularly advantageous when other routes of administration, suchas inhalation, would be difficult, for example when a patient issuffering from lung complications.

It will be recognized by those skilled in the art that the optimalquantity and spacing of individual dosages of a p38 MAPK inhibitor willbe determined by the nature and extent of the condition being treated,the form, route and site of administration, and the particular patientbeing treated, and that such optimums can be determined by conventionaltechniques. It will also be appreciated by one of skill in the art thatthe optimal course of treatment, i.e. the number of doses of a p38 MAPKinhibitor given per day for a defined number of days, can be ascertainedby those skilled in the art using conventional course of treatmentdetermination tests. However, in view of its key signalling role,targeting p38 MAP kinase may result in unwanted side effects. In orderto minimise any such side effects, the p38 MAPK inhibitor may beadministered to a patient in accordance with the present invention for amaximum period of 1-5 days, preferably 1-3 days. In some embodiments,the p38 MAPK inhibitor may be administered for just one or two days.Once-a-day treatment may also be preferred to minimise any deleteriousside effects.

In yet another aspect of the present invention there is provided apharmaceutical composition for use in the treatment or prevention ofhypercytokinemia in a human patient, the composition comprising a p38MAPK inhibitor having Formula I, Formula II and/or Formula III,pharmaceutically acceptable salts or solvates thereof.

In particular, there is provided a pharmaceutical composition for use inthe treatment or prevention of severe influenza in a human patient, thecomposition comprising a p38 MAPK inhibitor having Formula I, Formula IIand/or Formula III, and pharmaceutically acceptable salts or solvatesthereof, optionally in combination with one or more pharmaceuticallyacceptable diluents or carriers. Diluents and carriers may include thosesuitable for parenteral, oral, topical, mucosal and rectaladministration.

A pharmaceutical composition comprising a p38 MAPK inhibitor (forexample a p38 MAPK inhibitor of Formula I, Formula II and/or FormulaIII, or pharmaceutically acceptable salts or solvates thereof), mayfurther comprise, or be administered in combination with, a furtheragent. The further agent may be an antimicrobial agent, or anticanceragent. In particular, the further agent may be an antiviral agent andmore specifically oseltamivir or a pharmaceutically acceptable saltthereof (e.g. oseltamivir phosphate).

As used herein, the term “pharmaceutically-acceptable salts or solvates”refers to salts or solvates that retain the desired biological activityof the subject compound and exhibit minimal undesired toxicologicaleffects. These pharmaceutically-acceptable salts may be prepared in situduring the final isolation and purification of the compound, or byseparately reacting the purified compound in its free acid or free baseform with a suitable base or acid, respectively.

The compounds of the invention may exist in crystalline ornon-crystalline (amorphous) form, or as a mixture thereof. For compoundsof the invention that are in crystalline form, the skilled artisan willappreciate that pharmaceutically-acceptable solvates may be formedwherein solvent molecules are incorporated into the crystalline latticeduring crystallization. Solvates may involve non-aqueous solvents suchas ethanol, isopropanol, DMSO, acetic acid, ethanolamine, and ethylacetate, or they may involve water as the solvent that is incorporatedinto the crystalline lattice. Solvates wherein water is the solvent thatis incorporated into the crystalline lattice are typically referred toas “hydrates”. Hydrates include stoichiometric hydrates as well ascompositions containing viable amounts of water. The invention includesall such solvates.

Certain compounds of the invention that exist in crystalline form,including the various solvates thereof, may exhibit polymorphism (i.e.the capacity to occur in different crystalline structures). Thesedifferent crystalline forms are typically known as “polymorphs”. Theinvention includes all such polymorphs. Polymorphs have the samechemical composition but differ in packing, geometrical arrangement, andother descriptive properties of the crystalline solid state. Polymorphs,therefore, may have different physical properties such as shape,density, hardness, deformability, stability, and dissolution properties.Polymorphs typically exhibit different melting points, IR spectra, andX-ray powder diffraction patterns, which may be used for identification.Different polymorphs may be produced, for example, by changing oradjusting the reaction conditions or reagents, used in making thecompound. For example, changes in temperature, pressure, or solvent mayresult in polymorphs. In addition, one polymorph may spontaneouslyconvert to another polymorph under certain conditions.

The pharmaceutical composition of the invention may be prepared e.g. forparenteral, subcutaneous, intramuscular, intravenous, intra-articular orperi-articular administration, particularly in the form of liquidsolutions or suspensions; for oral administration, particularly in theform of tablets or capsules; for topical e.g. pulmonary or intranasaladministration, particularly in the form of powders, nasal drops oraerosols and transdermal administration; for mucosal administration e.g.to buccal, sublingual or vaginal mucosa, and for rectal administratione.g. in the form of a suppository.

The composition may conveniently be administered in unit dosage form andmay be prepared by any of the methods well-known in the pharmaceuticalart, for example as described in Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Company, Easton, Pa., (1985). A formulationfor parenteral administration may contain as excipients sterile water orsaline, alkylene glycols such as propylene glycol, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, hydrogenatednaphthalenes and the like. A formulation for nasal administration may besolid and may contain excipients, for example, lactose or dextran, ormay be aqueous or oily solutions for use in the form of nasal drops ormetered spray. For buccal administration typical excipients includesugars, calcium stearate, magnesium stearate, pregelatinated starch, andthe like.

In particular, the pharmaceutical composition may be formulated for oraladministration. A composition suitable for oral administration maycomprise one or more physiologically compatible carriers and/orexcipients and may be in solid or liquid form. Tablets and capsules maybe prepared with binding agents, for example, syrup, acacia, gelatin,sorbitol, tragacanth, or poly-vinylpyrollidone; fillers, such aslactose, sucrose, corn starch, calcium phosphate, sorbitol, or glycine;lubricants, such as magnesium stearate, talc, polyethylene glycol, orsilica; and surfactants, such as sodium lauryl sulfate. A liquidcomposition may contain conventional additives such as suspendingagents, for example sorbitol syrup, methyl cellulose, sugar syrup,gelatin, carboxymethyl-cellulose, or edible fats; emulsifying agentssuch as lecithin, or acacia; vegetable oils such as almond oil, coconutoil, cod liver oil, or peanut oil; preservatives such as butylatedhydroxyanisole (BHA) and butylated hydroxytoluene (BHT). A liquidcomposition may be encapsulated in, for example, gelatin to provide aunit dosage form.

A solid oral dosage form may include tablets, two-piece hard shellcapsules and soft elastic gelatin (SEG) capsules.

A dry shell formulation typically comprises of about 40% to 60%concentration of gelatin, about a 20% to 30% concentration ofplasticizer (such as glycerin, sorbitol or propylene glycol) and about a30% to 40% concentration of water. Other materials such aspreservatives, dyes, opacifiers and flavours also may be present. Theliquid fill material may comprise a solid drug that has been dissolved,solubilized or dispersed (with suspending agents such as beeswax,hydrogenated castor oil or polyethylene glycol 4000) or a liquid drug invehicles or combinations of vehicles such as mineral oil, vegetableoils, triglycerides, glycols, polyols and surface-active agents.

In some embodiments, compositions of the invention may be administeredtopically to the lung. In some embodiments, therefore the composition ofthe invention may comprise a p38 MAPK inhibitor optionally incombination with one or more topically acceptable diluents or carriers.Topical administration to the lung may be achieved by use of an aerosolformulation. Aerosol formulations typically comprise the activeingredient suspended or dissolved in a suitable aerosol propellant, suchas a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC). Suitable CFCpropellants include trichloromonofluoromethane,dichlorotetrafluoromethane, and dichlorodifluoromethane. Suitable HFCpropellants include tetrafluoroethane (HFC-134a) and heptafluoropropane(HFC-227). The propellant typically comprises 40% to 99.5% e.g. 40% to90% by weight of the total inhalation composition. The formulation maycomprise excipients including co-solvents (e.g. ethanol) and surfactants(e.g. lecithin, sorbitan trioleate and the like). Aerosol formulationsare packaged in canisters and a suitable dose is delivered by means of ametering valve (e.g. as supplied by Bespak, Valois or 3M).

Topical administration to the lung may also be achieved by use of anon-pressurised formulation such as an aqueous solution or suspension.This may be administered by means of a nebuliser. Topical administrationto the lung may also be achieved by use of a pressured metered doseinhaler (pMDI) or a dry-powder formulation. A dry powder formulationwill contain the p38 MAPK inhibitor in finely divided form, typicallywith a mass mean diameter (MMAD) of 1-10 microns. The formulation willtypically contain a topically acceptable diluent such as lactose,usually of large particle size e.g. a mass mean diameter (MMAD) of 100μm or more. Example dry powder delivery systems include SPINHALER,DISKHALER, TURBOHALER, DISKUS and CLICKHALER.

In yet another aspect of the present invention there is provided amethod for treating or preventing hypercytokinemia in a human patient inneed thereof comprising administering to the patient a therapeuticallyor prophylactically effective amount of a p38 MAPK inhibitor havingFormula I, Formula II or Formula III or pharmaceutically acceptablesalts or solvates thereof. In particular, there is a provided a methodfor treating or preventing severe influenza virus infection.

The method may include administering a p38 MAPK inhibitor in combinationwith a further agent. The further agent may be an antimicrobial agent,or anticancer agent. In particular, the further agent may be anantiviral agent and more specifically oseltamivir or a pharmaceuticallyacceptable salt thereof (e.g. oseltamivir phosphate). In particular, thep38 MAPK inhibitor may be administered orally.

It will be understood that the description of the present inventionherein insofar as it relates to p38 MAP kinase inhibitors for use in thepresent invention (for example for use in the treatment or prevention ofhypercytokinemia (e.g. hypercytokinemia associated with severe influenzavirus infection) and/or for use in the treatment or prevention of severeinfluenza) is applicable equally to the various aspects of the inventionset forth herein that pertain to methods of treatment of the inventionin humans and animals (for example methods of treatment or prevention ofhypercytokinemia (e.g. hypercytokinemia associated with severe influenzavirus infection) in a subject (e.g. a human patient) in need thereofand/or methods of treatment or prevention of severe influenza in asubject (e.g. a human patient) in need thereof)).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the identification of signalling pathways.

FIG. 2 illustrates the mapping the activity of genes in IL-1, TNFα andIL-6 stimulated pathways. These cytokines are produced by influenzavirus infected cells and found to be increased in the blood of peopleinfected with influenza, particularly those hospitalised with severeinfluenza. The hatched lines indicate gene expression levels with linessloping from top right to bottom left indicating upregulation (e.g.TNF-α), and lines sloping from top left to bottom right (e.g. LBP)indicating down-regulation; genes that are both upregulated anddown-regulated are indicated by cross-hatching. The line spacingrepresents the intensity of up- or down-regulation, with more denselypacked lines indicating greater activity. The maps were generated usingIPA. A “route” through a pathway is defined as a single contiguousconnection of proteins that extend from the plasma membrane through tothe nucleus.

FIG. 3 shows an example of scoring a route through the IL-6 pathway ofFIG. 2.

FIG. 4 shows three key cell types involved in the pathology of severeinfluenza.

FIG. 5 is a schematic of influenza ‘soup’ production andelectrogenerated chemiluminescence analysis in human epithelial cells.Infected cell “soups” contain key cytokines found in clinical samples.

FIG. 6 shows Western blotting of phosphorylated P38 and HSP27 inresponse to viral soup application and effects of P38 inhibition inepithelial cells using the p38 MAPK inhibitor PH 797804.Electrogenerated chemiluminescence analysis of inflammatory cytokineproduction in response to viral soup application, and effects of P38inhibition in epithelial cells.

FIG. 7 shows Western blotting of phosphorylated HSP27 in response toviral soup application and effects of P38 inhibition in endothelialcells. Electrogenerated chemiluminescence analysis of inflammatorycytokine production in response to viral soup application, and effectsof P38 inhibition in endothelial cells.

FIG. 8 shows Western blotting of phosphorylated HSP27 in response toviral soup application and effects of P38 inhibition in immune cells.Electrogenerated chemiluminescence analysis of inflammatory cytokineproduction in response to viral soup application, and effects of P38inhibition in immune cells. Cell viability of immune cells in responseto increasing concentrations of P38 MAPK inhibitor.

FIG. 9 shows electrogenerated chemiluminescence analysis of inflammatorycytokine production in response to LPS, CD-3 and viral soup application.Effects of P38 inhibition on viral soup induced inflammatory cytokine inimmune cells.

FIG. 10 shows the effects of p38 and MEK inhibitors on IP10 productionby HUVECs stimulated with 70% HBEC viral soup. Secreted cytokine levelswere assayed by electrogenerated chemiluminescence. Statisticalsignificance was calculated using one-way ANOVA with Dunnett's multiplecomparison post hoc test.

FIG. 11 shows compound effects on IL-1b production from immune cells.Each point on the dot plot represents an individual experiment.Statistical significance was calculated using one-way ANOVA withDunnett's multiple comparison post hoc test.

FIG. 12. shows compound effects on TNFa production from immune cells.Each point on the dot plot represents an individual experiment.Statistical significance was calculated using one-way ANOVA withDunnett's multiple comparison post hoc test.

FIG. 13 shows compound effects on IP10 production from endothelialcells. Each point on the dot plot represents an individual experiment.Statistical significance was calculated using one-way ANOVA withDunnett's multiple comparison post hoc test.

FIG. 14 shows compound effects on IL8 production from endothelial cells.Each point on the dot plot represents an individual experiment.Statistical significance was calculated using one-way ANOVA withDunnett's multiple comparison post hoc test.

FIG. 15 shows the cytokine levels in 28 healthy volunteers infected withinfluenza virus (samples collected at Day −1 to Day 28, hollow dots) and30 individuals hospitalised with severe influenza (stippled dots). Bloodsamples from the severe patients were collected between 24 and 72 hoursafter admission to hospital. The statistical significance of differencesin cytokine levels between the infected healthy volunteers (D2 and D3)and the hospitalised patients (influenza A or B) was calculated usingthe Mann-Whitney U test.

FIG. 16 shows the effect of HBEC viral soup on inflammatory mediatorproduction in HBEC cells measured by MSD analysis and the inhibitoryeffect of the compound of Formula III.

FIG. 17 shows inflammatory cytokine production by HUVEC cells treatedwith either TNFa plus IL-6, or HBEC viral soup as measured by MSDanalysis in endothelial cells and the inhibitory effect of the compoundof Formula III.

FIG. 18 shows inflammatory cytokine production in immune cells treatedwith either TNFa plus IL-6, or A549 viral soup as measured by MSDanalysis in immune cells and the inhibitory effect of the compound ofFormula III.

FIG. 19 shows the effect of combining p38 MAPK inhibitors withoseltamivir.

FIG. 20 shows inflammatory cytokine production by HUVEC cells treatedwith either TNFa plus IL-6, or HBEC viral soup as measured by MSDanalysis in endothelial cells and the inhibitory effect of the compoundof Formula II.

FIG. 21 shows inflammatory cytokine production in immune cells treatedwith either TNFa plus IL-6, or A549 viral soup as measured by MSDanalysis in immune cells and the inhibitory effect of the compound ofFormula II.

FIG. 22 shows the effect of combining the compound of Formula II withoseltamivir on the anti-viral properties of oseltamivir.

FIG. 23 shows the effect of combining oseltamivir the compound ofFormula II on the anti-inflammatory properties of the compound ofFormula II.

EXAMPLES Example 1 Identification of p38 MAPK by Transcriptomic Analysis

Bioinformatics analysis of transcriptomic data from blood samplescollected from human volunteers and patients infected with influenza wasused to map the signalling pathways activated in the human host responseto influenza infection in both uncomplicated (mild and moderate) andsevere influenza (see PHE Guidance on Use of Anti-Viral Agents for the

Treatment and Prophylaxis of Seasonal Influenza (2015-16), version 6.0,September 2015). Human viral challenge studies were carried out andtranscriptomic data from those studies were used for mapping the former,while transcriptomic data from a field-based sampling study (Hoang, L.T. et al., 2014) were used for mapping the latter. Comparison ofsignalling pathways identified by comparing both datasets enabled theidentification of signalling pathways that are very active in severeinfluenza versus mild and moderate influenza. Further analysis ofindividual pathway components identified p38 MAPK as a key “node” in anumber of these active pathways.

Healthy human volunteers were intranasally challenged with influenzaA/Wisconsin/67/2005 (H3N2) (Zaas, A. K. et al., Gene expressionsignatures diagnose influenza and other symptomatic respiratory viralinfection in humans, Cell Host Microbe, 2009; 17: 207- 217 andDavenport, E. E. et al., Transcriptomic profiling facilitatesclassification of response to influenza challenge, J. Mol. Med., 2015;93: 105-114) or with influenza A/Perth/16/2009 (H3N2) (internal study,not published). PAXgene™ samples of whole blood were collected from thevolunteers at various time points for subsequent transcriptome analysis.Methods for influenza A/Wisconsin/67/2005 (H3N2) viral challenge, casedefinitions, sample collection, RNA purification and microarray analysisare as detailed in Zaas et al., 2009 and Davenport et al., 2015. Methodsfor influenza A/Perth/16/2009 (H3N2) viral challenge, case definitionsand sample collection were as described for the Wisconsin strain exceptRNA purification and microarray analysis using Affymetrix HGU133 Plus2.0 arrays were performed by Almac(https://web.archive.org/web/20160317153848/http://www.almacgroup.com/).Methods for recruiting patients with severe influenza, blood samplecollection, RNA purification and microarray analysis are as detailed inHoang et al., 2014.

Microarray data files for the Zaas et al., 2009, Davenport et al., 2015and Hoang et al., 2014 studies were downloaded from the Gene ExpressionOmnibus (GEO) database(https://web.archive.org/web/20160622040853/http://www.ncbi.nlm.nih.gov/geo/)using the accession numbers GSE52428, GSE61754 and GSE61821,respectively. Microarray data (.CEL) files for the unpublished studywere downloaded from Almac and stored locally for bioinformaticsanalysis. All four transcriptomic datasets were processed and analysedusing the R (version 3.0.2.) integrated suite of software facilities fordata manipulation, calculation and graphical display(https://web.archive.org/web/20160623011408/http://www.R-project.org).Quality assessment of raw microarray data was performed usingstatistical methods standard in the art (e.g. Heber, S. and Sick, B.,Quality assessment of affymetrix genechip data, Omics, 2006; 10:358-368). Affymetrix datasets were normalised using the RobustMulti-array Average (RMA) method[https://www.bioconductor.org/packages/3.3/bioc/manuals/affy/man/affy.pdf]and Illumina datasets were normalised using the Lumi package[https://www.bioconductor.org/packages/3.3/bioc/manuals/lumi/man/lumi.pdf].Both packages were executed in the R environment. To facilitateannotation of probe-sets and gene names, Affymetrix chip definitionfiles (version 17.1.0) were downloaded from the BrainArray website(https://web.archive.org/web/2016062311275820160623112758/http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/17.1.0/ensg.asp)and Illumina chip definition files (illuminaHumanv4.db) were downloadedfrom the Bioconductor website(https://web.archive.org/web/20151209032754/http://bioconductor.org/packages/release/data/annotation/html/illuminaHumanv4.db.html).

The latter files were used with microarray data from Davenport et al.,2015 and Hoang et al, 2014.

Normalised Zaas et al., 2009, Davenport et al., 2015 and Perth datasetswere individually merged with the Hoang et al., 2014 dataset using theCOMBAT module in the InSilicoMerging package in Bioconductor(https://web.archive.org/web/20150905151657/http://www.bioconductor.org/packages/release/bioc/html/inSilicoMerging.html). Differential gene expressionanalysis on merged data sets was carried out using the limma package inR (https://www.bioconductor.org/packages/3.3/bioc/vignettes/limma/inst/doc/usersguide.pdf). For pairwise comparisons only data frominfected volunteers in the Zaas et al., 2009, Davenport et al., 2015 andPerth datasets were used, equating to 11, 14 and 5 subjects,respectively. From the Hoang et al., 2014 dataset only data for thethree H3N2-infected severe influenza patients in the data set were used.For each merged data set two pairwise comparisons were performed toidentify genes that were upregulated relative to baseline levels afterinfection with virus and then further upregulated in the severe patientsamples:

Perth: day −1 vs day 3 and; day 3 vs Hoang et al., 2014 severe.

Zaas et al., 2009: day -1 vs 60 hours and; 60 hours vs Hoang et al.,2014 severe.

Davenport et al., 2015: day 0 vs 48 hours and; 48 hours vs Hoang et al.,2014 severe.

To maximise the number of upregulated genes that could be mapped topathways, all genes showing fold-changes>0 were identified. Each of the6 resulting gene lists were analysed through the use of QIAGEN'sIngenuity® Pathway Analysis (IPA®, QIAGEN Redwood City,https://web.archive.org/web/20131021061639/http://www.ingenuity.com/).This resulted in the identification of 650 signalling pathways whichwere reduced to 353 after the removal of 297 metabolic pathways. FIG. 1summarises this process.

In order to interrogate the relevance of each of these signallingpathways to the pathogenesis of severe influenza, a manual scoringapproach was devised to identify very active “routes” within thesepathways in the complicated versus the “uncomplicated” influenzadatasets. In this context “routes” are defined as contiguous connectionsof proteins in a canonical pathway that extend from the plasma membranethrough to the nucleus. As a result, a canonical pathway may have anumber of different routes through it. Using this scoring approach,routes within IPA canonical pathways were mapped directionally from theplasma membrane to the nucleus and the ‘overlay’ function in IPA wasused to show gene activity. To illustrate this process an example ofthree pathways identified using this method is shown in FIG. 2.

Individual routes in the identified 353 pathways were manually scoredfor gene activity as exemplified in FIG. 3 for a route through the IL-1canonical pathway in which:

TABLE 1 Nodes Nodes not Pathway Route Up-regulated up-regulated IL-6IL6R + GP130- 81% SHC-Ras signalling SHC-GRB2-SOS- Ras-cRAF-MEK-ERK-ELK + SRF

In all, 491 routes showing >75% up-regulated nodes were identified(exemplified in Table 2 below). Of these, 95 routes containing >3 nodeswere identified in which 100% of all the nodes in the route wereupregulated in the Hoang et al., 2014 severe influenza dataset versusbaseline (D-1 or DO) in the Zaas et al., 2009, Davenport et al., 2015and Perth datasets (Table 3). Twenty-four of these 95 routes were shownto be >75% upregulated compared with pathways derived from the mild andmoderate H3N2 and H1N1 influenza datasets from Hoang et al., 2014 (H3N2and H1N1—mild and moderate) and Zaas et al., 2009 (H1N1-D-1 and 60 h;Table 4).

Inspection of the 95 routes highlighted a number of potentiallytargetable nodes from which p38MAPK was chosen because of its wellcharacterised role in inflammation and the availability of high qualityclinically tested small molecule inhibitors for use in in vitro and exvivo studies.

TABLE 2 An example of route scoring analysis. Nodes↑ Pathway Route Nodes(%) ↓Nodes NFKB growth factor receptors-RAS- 10 92 IKKBRAF-MEKK1-IKKa-NFKB2- RELB-lymphogenesis NFKB IL-1R/TLR-MYD88-TYRAP- 893 IKKB IRAP-IRAK-TRAF6-TAK1- IKKa-IKBP65-P65NFKB- P65NFKB-inflammationRole of IL21Ralpha/IL2Rgamma-JAK3- 4 100 JAK1 and STAT1/3/5 JAK3 incytokine signalling PI3K-AKT Integrin-PINCH-ILK-PI3K-PP2A- 9 89signalling AKT-CRAF-MEK1/2-ERK1/2- RTK P70S6K-cell growth PI3K-AKTIntegrin-PINCH-ILK-PI3K-PP2A- 9 89 signalling AKT-CRAF-MEK1/2-ERK1/2-RTK P70S6K-cell growth PI3K-AKT Integrin-PINCH-ILK-PI3K-PP2A- 9 78signalling AKT-CRAF-MEK1/2-ERK1/2- integrin P70S6K-cell growth NFKBTNF-TANK-TRAF-FADD-RIP- 7 87.5 TNF- MAP3K3-IKKa-IKBP65- R/P65NFKB-P65NFKB- IKKB inflammation CNTF (CNTFR-LIFR-GP130)-JAK1/2- 10 90SHP2 signalling SHP2-GRB2-SOS-RAS-CRAF- MEK1/2-ERK1/2-P( )RSK-geneexpression CNTF (CNTFR-LIFR-GP130)-TYK2- 3 100 signalling STAT1/3-geneexpression role of JAK (GP130-OSMR)-intermediate 4 100 in IL-6 typesignalling-ERK1/2-p38MAPK- cytokine JNK-signalling signalling role ofJAK (GP130-OSMR)-JAK2- 3 100 in IL-6 type STAT1/3/5-gene expressioncytokine signalling role of JAK (GP130-OSMR)-STAT1/3-gene 2 100 in IL-6type expression cytokine signalling HER-2 (HER1/HER2)-GRB2-SOS-RAS- 5 80HER1- signalling (CYCLIND1-CDK6-CYCLINE- HER2 in breast p27KIP1)-cellcycle progression cancer and proliferation HER-2 (HER1/HER2)-PI3K-AKT- 475 HER1- signalling CYCLIND1-cell cycle progression HER2 in breastcancer role of (IFNAR1-IFNAR2)-TYK2- 4 100 JAk1, STAT2-STAT1-geneexpression JAK2 and TYK2 in interferon signalling IL-9(IL-9R-IL2R)-JAK3-IRS1/2-PI3K- 5 100 signalling PI3ksignalling

TABLE 3 Ninety-five routes containing 100% upregulated nodes in theHoang et al., 2014 severe influenza dataset versus the Zaas et al.,2009, Davenport et al., 2015 an dPerth baseline datasets. Number PathwayRoute of Nodes Acute myeloid FLT3-GRB2-SOS-RAS-RAF-MEK- 7 leukemiaERK1/2-cell proliferation signalling Gaq signallingGqR-Ga/b/y-PYK2-PI3K-AKT-IKK- 7 NFkB p38 MAPK TNFR/fas-TRADD/FAD-TRAF2-7 signalling Ask1-MKK4-P38MAKa-CHOP- transcription p38 MAPKTNFR/fas-TRADD/FAD-TRAF2- 7 signalling Ask1-MKK4-P38MAKa-ELK1-transcription p38 MAPK TNFR/fas-TRADD/FAD-TRAF2- 7 signallingAsk1-MKK4-P38MAPKa-MEF2 SAPK/JNK TRADD/RIP/FADD-TRAF2-GCKs- 7 signallingMEKK1-MKK4/7-JNK-ELK-1 Sertoli cell sertoli CLDN-ZO2-factin-actininalpha- 7 cell junction tubulin-KEAP1-Myo7a-junction signalling dynamicsHIF1a signalling RTK-PI3K-AKT-HIF1a-ARNT-ET1- 6 vascular tone HIF1asignalling RTK-PI3K-AKT-HIF1a-ARNT- 6 MMPs-ECM regulation IL6 signallingTNFR-TRAF2-TAK1-MKK4/7-JNK- 6 ELK1 Protein Kinase APKAr/PKAc-RAP1-BRAF-MEK1/2- 6 signalling ERK1/2-ELK1 SAPK/JNKTRADD/RIP/FADD-TRAF2-ASK1- 6 signalling MKK4/7-JNK-ELK1 ERK5 signallingSRC-MEKK2/3-MEK5-ERK5-SAP1 5 Glucocorticoid CYTOKINE RECEPTOR-TRAF2- 5Receptor TAK1-MKK4/7-P38MAPK- signalling STABILIZATION OF MRNA,TRANSLATION Growth Hormone GHR-JAK2-ERK1/2-CEBPA 5 signalling GrowthHormone GHR-JAK2-ERK1/2-P90RSK- 5 signalling SRF/ELK1 HIF1a signallingRTK-PI3K-AKT-HIF1a-ARNT- 5 GLUT HIF1a signallingRTK-PI3K-AKT-HIF1a-ARNT- 5 VEGF IL-22 signallingIL22R1/2-TYK2-STAT1/3/5-SOCS3 5 IL-8 CXCR1/2-PI3K-Akt-AP1- 5IntegrinAlphavBeta3 (Chemotaxis) IL-8 CXCR1/2-Ras-Raf-MEK1/2-ERK1/2- 5(Neutrophil Degranulation) leptin signalling in LEPR-JAK2-STAT3-(SOCS3-5 obesity POMC)-aMSH-anorexia Paxillin signallingIntegrina/b-FAK-GRB2-SOS-Ras- 5 ERK/MAPK Role of RIG likedsRNA-RIG1-IPS1-TRAF3-TBK1- 5 receptors in IRF7-(IFNa-MDA5/LGP2/RIG1)antiviral innate immunity Role of RIG like MDA5-IPS1-TRAF3-TBK1-IRF7- 5receptors in (IFNa-MDA5/LGP2/RIG1) antiviral innate immunity Role of RIGlike TRIM25-RIG1-IPS1-TRAF3-IRF7- 5 receptors in (IFNa-MDA5/LGP2/RIG1)antiviral innate immunity CD40 signalling CD40-JAK3-STAT3-ICAM1 4ceramide signalling EDG-SPHK-NFKB-AP1-activation of 4 inflammatory genesceramide signalling SMPD-(ceramide)-PI3K-AKT- 4 apoptosis EicosanoidPLA2-ALOX5-LTA4h-LTB4R- 4 signalling chemotaxis/proliferation/allergicasthma/angiogenesis/ G alpha I signalling GiCOUPLED RECEPTOR- 4Galphai/Gbeta/Ggamma-SRC-STAT3 Germ Cell-SertoliTGFbetaR-RAS-MEK1/2-ERK1/2- 4 Cell actin Junction signallingdepolymerisation GM-CSF signalling GMCSFRA-HCK-PI3K-AKT-cell 4survival/cell proliferation GM-CSF signalling GMCSFRA-JAK2-STAT3-(BCLXL-4 CYCLIND1) G-Protein Coupled Gicoupled receptor-GALPHAi/0- 4 ReceptorSRC-STAT3 signalling IGF-1 signalling IGF1R-JAK 1/2-STAT3-SOCS3 4 IL-8CXCR1/2-JNK-NFkB-ICAM-1 4 IL-8 CXCR1/2-PI3K-MEK1/2-ERK1/2- 4 (NeutrophilDegranulation) IL-8 CXCR1/2-Rho-NFkB-ICAM-1 4 JAK/STAT cytokinereceptor-JAK-STAT-(CFOS- 4 IL6-SOCS-BCLXL) MSP-RON RON-PI3K-PKCzeta-F-ACTIN- 4 signalling phagocytic activity in macrophages pathwayPI3K signalling in IL4R-IRS-P85/PI3K-P110/PI3K- 4 B Lymphocytes NFKBPPARα/RXRα ADIPOR-AMPK-P38MAPK- 4 Activation PPARalpha Production ofnitric TLR2/4-PI3K-AKT-NFKB-Inos 4 oxide and ROS in macrophagesProduction of nitric TLR2/4-MKK4/-JNK-AP1 4 oxide and ROS in macrophagesRAR activation IL-3Ra/b-JAK2-STAT5-RAR/RXR 4 Role of MAPKASK-1-MKK4/7-JNK-CASP3- 4 signalling in the APOPTOSIS Pathogenesis ofInfluenza Role of RIG like dsRNA-RIG1-IPS1-TRAF3-IRF7- 4 receptors in(IFNa-MDA5/LGP2/RIG1) antiviral innate immunity Role of RIG likeMDA5-IPS1-TRAF3-IRF7-(IFNa- 4 receptors in MDA5/LGP2/RIG1) antiviralinnate immunity signalling by Rho Integrin-ARHGEF-RHO-FAK- 4 FamilyGTPases cytoskeletal reorganisation signalling by RhoIntegrin-ARHGEF-RHO-PKNI-cell 4 Family GTPases traffickingSphingosine-1- SIPR(2/3/4)-GAI-PI3K-AKT-CELL 4 phosphate SURVIVALsignalling Tec Kinase Integrin-FAK-TEC KINASE- 4 signalling (FAK, PKC,PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT- TFII-1) Tec KinaseTCR-SRC-TEC KINASE- 4 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE,NFKB, JNK, STAT- TFII-1) Acute myeloid FLT3-STAT3/5-PIM1-regulates 3leukemia signalling apoptosis Antioxidant actionCSF2Ralpha/beta-JAK2-STAT5-gene 3 of vitamin C expression CNTFsignalling (CNTFR-LIFR-GP130)-TYK2- 3 STAT1/3-gene expression DendriticCell LTbetaR-IKK-RELB/NFKB-cross 3 Maturation presentation EPHRINEPHA-JAK2-STAT3-CELL 3 RECEPTOR PROLIFERATION signalling EPHRINEPHB-PI3KG-AKT-CELL 3 RECEPTOR MIGRATION, CELL signalling PROLIFERATIONEPHRIN INTEGRIN-MEK1/2-ERK1/2-AXON 3 RECEPTOR GUIDANCE, CELL signallingPROLIFERATION FcyRIIB signalling FCyR-BTK-JNK-apoptosis 3 in Blymphocytes Glucocorticoid CYTOKINE RECEPTOR-JAK2- 3 Receptor signallingSTAT1 Glucocorticoid CYTOKINE RECEPTOR-JAK3- 3 Receptor signallingSTAT3/5 GNRH signalling GnRHR-Gai-NfkB 3 IL-12 signallingTLR4-p38/MAPK-IL12 3 and Production in Macrophages IL-3 signallingIL3Ralpha/beta-JAK1/2- 3 STAT1/3/5/6-gene expression IL6 signallingGP130 (IL6R)-JAK2-STAT3-gene 3 expression IL-8 CXCR1-PLD-NADPH oxidase-3 (Superoxide production—Respiratory Burst) IL-8 CXCR1-G Proteinalpha/beta/gamma- 3 PI3Ky-(Chemotaxis—Respiratory Burst) LPS stimulatedTLR4-IKK-IKB-NFKB-gene 3 MAPK signalling expression mTOR signallingNutrients-RHEB-mTORc2-AKT- 3 PI3K/AKT signalling mTOR signallingNutrients-RHEB-mTORc2-AKT- 3 Rho/PKC)-actin organisation PDGF signallingPDGFRa/b-SPHK-CRK-mitogenesis 3 Protein Kinase A PKA-PHK-PYG-glycolysis3 signalling Regulation of CNG-CALPAIN-RB 3 cellular mechanics bycalpain protease role of JAK in IL-6 (GP130-OSMR)-JAK2-STAT1/3/5- 3 typecytokine gene expression signalling Role of JAK2 inGHR-JAK2-IRS-PI3K/AKT 3 Hormone-like SIGNALLING cytokine signalling Roleof JAK2 in GHR-JAK2-STAT1/3-GENE 3 Hormone-like EXPRESSION cytokinesignalling Role of JAK2 in GHR-JAK2-STAT5-GENE 3 Hormone-like EXPRESSIONcytokine signalling Role of GP130-JAK2-STAT3-gene 3 Macrophages,expression Fibroblasts and Endothelial Cells in Rheumatoid ArthritisRole of Pattern NALP3-casp1-IL1b 3 Recognition Receptors in Recognitionof Bacteria and Viruses Role of Pattern NOD1-Casp1-IL1b 3 RecognitionReceptors in Recognition of Bacteria and Viruses Role of PI3K/AKTPI3K-AKT-IKB, NFKB 3 signalling in the Pathogenesis of Influenza Role oftissue factor PAR2-ERK1/2-HBEGF-angiogenesis 3 in cancer Role of tissuefactor PAR2-ERK1/2-VEGFa-angiogenesis 3 in cancer Role of tissue factorPAR2-p38/MAPK-uPar-tumour 3 in cancer invasion Role of tissue factorPAR2-p38/MAPK-IL-1b- 3 in cancer angiogenesis Role of tissue factorPAR2-p38/MAPK-VEGFa- 3 in cancer angiogenesis STAT3 pathway cytokinereceptors-TYK2/JAK2- 3 STAT3-transcription-immuneresponse-proliferation-survival STAT3 pathway GFR-JAK2/SRC-STAT3- 3transcription-immune response- proliferation-survival Synaptic long termAMPAR-Lyn-PKC-Phosphorylation 3 depression Tec Kinase FCeR1-TEC kinase-3 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT-TFII-1) Tec Kinase TLR4-TEC kinase- 3 signalling (FAK, PKC, PAK, VAV,FACTIN, RHOGTPASE, NFKB, JNK, STAT- TFII-1)

TABLE 4 Comparison of route scores between H3N2 and H1N1. H3N2 Severe vsSevere vs Severe vs Severe vs Baseline Peak Mild Moderate Pathway RouteH3N2 H3N2 H3N2 H3N2 Growth Hormone GHR-JAK2-ERK1/2-CEBPA 100.00 93.33100.00 100 signalling PPARα/RXRα ADIPOR-AMPK-P38MAPK- 100.00 100.00100.00 100 Activation PPARalpha-REGULATION of growth hormone genesGM-CSF signalling GMCSFRA-HCK-PI3K-AKT-cell 100.00 100.00 100.00 100survival/cell proliferation Sphingosine-1- SIPR(2/3/4)-GAI-PI3K-AKT-100.00 100.00 100.00 100 phosphate signalling CELL SURVIVAL ceramidesignalling SMPD-(ceramide)-PI3K-AKT- 100.00 100.00 100.00 100 apoptosisIL-8 CXCR1/2 - PI3K-MEK1/2- 100.00 93.33 100.00 100 ERK1/2- (NeutrophilDegranulation) Paxillin signalling Integrina/b-FAK-GRB2-SOS - 100.00100.00 100.00 100 Ras-ERK/MAPK Tec Kinase FCeR1-TEC kinase- 100.00100.00 100.00 100 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE,NFKB, JNK, STAT-TFII-1) Tec Kinase TCR-SRC-TEC KINASE- 100.00 100.00100.00 100 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK,STAT-TFII-1) Tec Kinase TLR4-TEC kinase- 100.00 100.00 100.00 100signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK,STAT-TFII-1) signalling by Rho Integrin-ARHGEF-RHO-PKNI- 100.00 100.00100.00 100 Family GTPases cell trafficking Regulation of CNG-CALPAIN-RB100.00 100.00 100.00 100 cellular mechanics by calpain protease TecKinase Integrin-FAK-TEC KINASE- 100.00 100.00 100.00 100 signalling(FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT-TFII-1) Acutemyeloid FLT3-GRB2-SOS-RAS-RAF- 100.00 100.00 100.00 85.71 leukemiasignalling MEK-ERK1/2-cell proliferation signalling by RhoIntegrin-ARHGEF-RHO-FAK- 100.00 95.23 100.00 100 Family GTPasescytoskeletal reorganisation IL-8 CXCR1/2 - Ras-Raf-MEK1/2- 100.00 91.67100.00 100 ERK1/2- (Neutrophil Degranulation) JAK/STAT cytokinereceptor-JAK-STAT- 100.00 91.67 100.00 100 (CFOS-IL6-SOCS-BCLXL) MSP-RONsignalling RON-PI3K-PKC zeta- F-ACTIN- 100.00 91.67 100.00 100 pathwayphagocytic activity in macrophages Germ Cell-SertoliTGFbetaR-RAS-MEK1/2- 100.00 91.67 100.00 100 Cell Junction ERK1/2-actindepolymerisation signalling Role of MAPK ASK-1-MKK4/7-JNK-CASP3 - 100.0083.33 100.00 100 signalling in the APOPTOSIS Pathogenesis of InfluenzaRole of PI3K/AKT PI3K-AKT-IKB, NFKB 100.00 77.78 100.00 100 signallingin the Pathogenesis of Influenza Protein Kinase A PKAr/PKAc-RAP1-BRAF-100.00 100.00 83.33 83.33 signalling MEK1/2-ERK1/2-ELK1 IL-8 CXCR1/2 -PI3K-Akt-AP1- 100.00 80.55 80.00 100 IntegrinAlphavBeta3 (Chemotaxis)Production of nitric TLR2/4-MKK4/7-JNK-AP1 100.00 91.67 75.00 100 oxideand ROS in macrophages H1N1 Severe vs Severe vs Severe vs Severe vsBaseline Peak Mild Moderate Pathway Route H1N1 H1N1 H1N1 H1N1 GrowthHormone GHR-JAK2-ERK1/2-CEBPA 75 75 75 75 signalling PPARα/RXRαADIPOR-AMPK-P38MAPK- 100 100 100 75 Activation PPARalpha-REGULATION ofgrowth hormone genes GM-CSF signalling GMCSFRA-HCK-PI3K-AKT-cell 100 100100 100 survival/cell proliferation Sphingosine-1-SIPR(2/3/4)-GAI-PI3K-AKT- 100 100 100 100 phosphate signalling CELLSURVIVAL ceramide signalling SMPD-(ceramide)-PI3K-AKT- 100 100 100 100apoptosis IL-8 CXCR1/2 - PI3K-MEK1/2- 75 75 100 100 ERK1/2- (NeutrophilDegranulation) Paxillin signalling Integrina/b-FAK-GRB2-SOS - 100 100100 100 Ras-ERK/MAPK Tec Kinase FCeR1-TEC kinase- 100 100 100 100signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK,STAT-TFII-1) Tec Kinase TCR-SRC-TEC KINASE- 100 100 100 100 signalling(FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT-TFII-1) TecKinase TLR4-TEC kinase- 100 100 100 100 signalling (FAK, PKC, PAK, VAV,FACTIN, RHOGTPASE, NFKB, JNK, STAT-TFII-1) signalling by RhoIntegrin-ARHGEF-RHO-PKNI- 75 75 75 75 Family GTPases cell traffickingRegulation of CNG-CALPAIN-RB 100 100 100 100 cellular mechanics bycalpain protease Tec Kinase Integrin-FAK-TEC KINASE- 100 100 100 100signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK,STAT-TFII-1) Acute myeloid FLT3-GRB2-SOS-RAS-RAF- 100 100 100 100leukemia signalling MEK-ERK1/2-cell proliferation signalling by RhoIntegrin-ARHGEF-RHO-FAK- 100 100 100 100 Family GTPases cytoskeletalreorganisation IL-8 CXCR1/2 - Ras-Raf-MEK1/2- 80 80 100 100 ERK1/2-(Neutrophil Degranulation) JAK/STAT cytokine receptor-JAK-STAT- 75 75100 100 (CFOS-IL6-SOCS-BCLXL) MSP-RON signalling RON-PI3K-PKC zeta-F-ACTIN- 75 100 75 75 pathway phagocytic activity in macrophages GermCell-Sertoli TGFbetaR-RAS-MEK1/2- 100 100 100 100 Cell JunctionERK1/2-actin depolymerisation signalling Role of MAPKASK-1-MKK4/7-JNK-CASP3 - 75 75 75 75 signalling in the APOPTOSISPathogenesis of Influenza Role of PI3K/AKT PI3K-AKT-IKB, NFKB 100 100 7575 signalling in the Pathogenesis of Influenza Protein Kinase APKAr/PKAc-RAP1-BRAF- 100 83.33 83.33 83.33 signalling MEK1/2-ERK1/2-ELK1IL-8 CXCR1/2 - PI3K-Akt-AP1- 75 80 100 100 IntegrinAlphavBeta3(Chemotaxis) Production of nitric TLR2/4-MKK4/7-JNK-AP1 100 100 100 100oxide and ROS in macrophages

Example 2 Effects of p38MAPK Inhibition on Inflammatory Mediator Releasein Key Cell Types Relevant to Severe Influenza

Two p38 MAPK inhibitors were used in in vitro and ex vivoexperiments: 1) PH797804, an ATP-competitive highly potent, selectiveand metabolically stable inhibitor of p38 (Hope HR1, et al.,Anti-inflammatory properties of a novel N-phenyl pyridinone inhibitor ofp38 mitogen-activated protein kinase: preclinical-to-clinicaltranslation, J. Pharmacol. Exp. Ther., 2009 (December); 331(3): 882-95);and 2) Dilmapimod (SB-681323 -Betts JC1, et al., Gene expression changescaused by the p38 MAPK inhibitor Dilmapimod in COPD patients: analysisof blood and sputum samples from a randomized, placebo-controlledclinical trial, Pharmacol. Res. Perspect., 2015 (Feb); 3(1): e00094).The structures of these compounds are as follows:

Experimental testing of p38MAPK inhibition was carried out in three celltypes that are key players in the pathology of severe influenza:epithelial; endothelial; and immune cells (FIG. 4). In theseexperiments, cells were pre-treated with simple and/or complex stimuliin order to simulate the action of inflammatory mediators that areproduced from influenza-infected epithelial cells. In the case of theformer, tumour necrosis factor alpha (TNFa) plus interleukin 6 (IL-6)was used to stimulate endothelial and immune cells. In the case of thelatter, conditioned medium (viral “soup”) derived either from influenzavirus infected A549 cells (adenocarcinoma human alveolar basalepithelial cells), or primary human bronchial epithelial cells (HBECs)was used to stimulate epithelial, endothelial and immune cells.

Epithelial Cells

A549 cells (adenocarcinoma human alveolar basal epithelial cells) orHBEC (Human Bronchial Epithelial Cells) were infected withA/Perth/16/2009(H3N2) virus from which viral conditioned media (or‘viral soup’) was collected. In the experiments described here, theapplication of viral soup either back onto epithelial cells or onto theother cell types of interest (endothelial and immune) was performed inorder to simulate the action of inflammatory mediators that are producedfrom Influenza infected epithelial cells.

For infection, high titre stocks of Influenza (H3N2) virus were producedby infection of MDCK-RVL cells (available from ATCC as MDCK (NBL-2)(ATCC® CCL-34), derived from a kidney of an apparently normal adultfemale cocker spaniel, September, 1958, by S. H. Madin and N. B. Darby.The line is hyperdiploid, and there is a bi-modal chromosome numberdistribution. There are no consistent identifiable marker chromosomes.One normal X chromosome is present in most spreads. The cells arepositive for keratin by immunoperoxidase staining). MDCK-RVL cells wereplated in T175 flask and allowed to grow to 85-90% confluence. The nextday, cells were washed twice with infection media. The InfluenzaA/Perth/16/2009 (H3N2) stock was removed from −80° C. and thawed on ice.The cells were infected with virus stock at 0.01 MOI for one hour ininfection media. At the end of incubation period, unbound virus wasremoved from the cells. The cells were washed once with infection mediaand overlaid with infection media and allowed to incubate for 48 hoursin 37° C. at 5% CO₂/air incubator. After incubation, the flasks werefrozen at −80° C. for a day. Next day, the flasks were thawed at roomtemperature and virus supernatant was centrifuged (2000 g, 10 min) andpooled together. The virus stock was aliquoted and stored at −80° C.

Viral “soups” were prepared using the A549 cell line and primary humanbronchial epithelial cells (HBECs). For the preparation of A549 soupscells were plated in a T175 flask and allowed to grow to obtain 85-90%confluence. The cells were washed twice with infection media. TheInfluenza A/Perth/16/2009 (H3N2)-WGC stock was removed from −80° C. andthawed on ice. The cells were infected with virus stock at 0.01 MOI forone hour in infection media. The unbound virus was removed from thecells at the end of the incubation period and cells were washed oncewith infection media before overlaying with infection media. The cellswere incubated for 48 hours in 37° C. at 5% CO₂/air incubator. Afterincubation, the media (‘viral soup’) was collected from all flasks,centrifuged (2000 g, 10 min) and pooled together. The viral soup wasaliquoted and stored at −80° C. For the preparation of HBEC viral soupcells were cultured to passage P-3 or P-4 in Bronchial Epithelial CellGrowth Medium (BECGM; Lonza). For infection, cells were washed twicewith BECGM then infected with Influenza A/Perth/16/2009 (H3N2) virusstock at 0.01 MOI for one hour in infection medium (BECGM containing1.06 USP/NF units per ml TPCK trypsin). The cells were then overlaidwith infection medium and incubated for 48 hours at 37° C. in 5%CO₂/air. The HBEC viral soup was then processed in a similar way to A549soup.

Viral growth kinetics were optimised for generating viral soups. Todetermine multistep growth curves, A549 cells or HBECs were infectedwith virus at an MOI of 0.01 TCID₅₀/cell at 37° C. for one hour.Following incubation, the cells were washed and overlaid with respectiveinfection media. The samples were harvested for viral titres andmeasurement of cytokines at various time points for 72 hours. The viraltitres were obtained by TCID50 on MDCK cells and the presence ofinflammatory mediators was assessed by MSD chemiluminescence assay usingmethods as recommended by the vendor(https://web.archive.org/web/20160522190937/https://www.mesoscale.com/).

Prior to experimental testing both A549 and HBEC soup preparations wereevaluated by electrogenerated chemiluminescence for the presence ofinflammatory mediators using methods as recommended by the vendor(https://web.archive.org/web/20160522190937/https://www.mesoscale.com/).Both soup preparations were found to contain elevated levels of thefollowing cytokines (IL1-β, IL-6, IL-8, IL-10, TNFα and RANTES; FIG. 5).

In vitro data were generated from A549 cells to test the effect of A549viral soup application on p38 activation as measured by western blottingof the phosphorylation status of p38MAPK itself and downstreamsignalling target, HSP27. For western blotting confluent cells werewashed in PBS and lysed in RIPA with protease inhibitor (Sigma-P8340),phosphatase inhibitor cocktails 2 and 3 (Sigma P5726 and P0044) andphosphatase inhibitors Na₃VO₄ and NaF on ice. Protein concentrationswere determined using the Pierce BCA Protein Assay Kit and equalconcentrations of each sample created in 4× Laemmli sample buffer(BIO-RAD 161-0747) with 2-mercaptoethanol. Samples were run on a 12% geland then transferred on to nitrocellulose. Membranes were blocked using5% milk powder, then hybridised with p38 MAPK rabbit antibody (CellSignalling Technologies, cat. no. 9212S) and phosphorylated p38 MAPKrabbit antibody (Cell Signalling Technologies, cat. no. 9211S), or HSP27antibody (Cell signalling Technologies, cat. no. 2402) and phospho-HSP27antibody (Cell signalling Technologies, cat. no. 9709). Secondaryantibodies were anti-rabbit HRP (Cell signalling Technologies catalogueno. CS7074P2) and anti-mouse HRP (Cell signalling, catalogue no. 7076S),respectively. Membranes were stripped for re-probing using Restore™ PLUSWestern Blot Stripping Buffer (Life technologies # 46430). The membraneswere treated with Amersham ECL Prime Western Blotting Detection Reagent(GE/Amersham #RPN2232) and imaged using ChemiDoc™ Touch Imaging System(BIORAD). Analysis was performed using the Image Lab software.

Induction of phosphorylation on both of these enzymes was detectedindicating that p38MAPK is activated following application of A549 viralsoup (FIG. 6). Furthermore, incubation with p38 MAPK inhibitors(Dilmapimod and PH 797804) was shown to dose dependently inhibit theinduction of both p38MAPK and HSP27 phosphorylation by our A549 viralsoup, confirming that this induction is a p38MAPK dependent processwithin these epithelial cells.

The effect of A549 viral soup on inflammatory cytokine production inA549 cells as measured by electrogenerated chemiluminescence was alsoexplored. As shown in FIG. 6, A549 soup was found to induce theproduction of key inflammatory cytokines (IL-6 and IP-10) to a greaterextent compared with control uninfected A549 soup. Application of thetwo p38 MAPK inhibitors on A549 cells prior to A549 viral soupapplication significantly attenuated release of both IL-6 and IP-10(FIG. 6 shows the data for the p38 MAPK inhibitor PH 797804). These datademonstrate that the release of inflammatory mediators in response toA549 viral soup application on A549 cells is a p38MAPK dependentprocess.

Endothelial Cells

The application of HBEC viral soup on to Human Umbilical VeinEndothelial Cells (HUVECs) was performed in order to simulate theinteraction of inflammatory mediators that are produced from Influenzainfected epithelial cells onto endothelial cells.

In vitro data were generated from HUVEC cells to test the effect of HBECviral soup application on p38 activation as measured by western blottingof the phosphorylation status of the p38MAPK downstream signallingtarget, HSP27. Induction of phosphorylation on HSP27 was detectedindicating that p38MAPK is activated following application of HBEC viralsoup (FIG. 7). Furthermore, pre-incubation with p38 MAPK inhibitors(Dilmapimod and PH 797804) was shown to dose dependently inhibit theinduction of HSP27 phosphorylation by the HBEC viral soup, confirmingthat this induction is a p38MAPK dependent process within theseendothelial cells.

The effect of HBEC viral soup on inflammatory cytokine production inHUVEC cells as measured by electrogenerated chemiluminescence (seemethods) was also explored. As shown in FIG. 7, HBEC viral soup wasfound to induce the production of key inflammatory cytokines (IL-6 andIP-10) to a greater extent compared with control HBEC uninfected soup.Incubation of HUVEC cells with the two p38 MAPK inhibitors prior to HBECviral soup application was found to significantly attenuate the HBECviral soup induced release of both IL-6 and IP-10 (FIG. 7). These datademonstrate that the release of inflammatory mediators in response toHBEC viral soup application in HUVEC endothelial cell is a p38MAPKdependent process.

Immune Cells

The application of A549 viral soup onto human Peripheral BloodMononuclear Cells (PBMCs) was performed in order to simulate theinteraction of inflammatory mediators that are produced from Influenzainfected epithelial cells onto immune cells. PBMCs were isolatedaccording to the manufacturers recommendations (Boyum, A., Separation ofleucocytes from blood and bone marrow, Scand. J. Clin. Lab. Invest.,1968, 21, suppl. 97).

Ex vivo data were generated from immune cells to test the effect of A549viral soup application on p38 activation as measured by Western blotting(see above for method) of the phosphorylation status of the p38MAPKdownstream signalling target, HSP27. Induction of phosphorylation onHSP27 was detected, indicating that p38MAPK is activated followingapplication of A549 viral soup (FIG. 8). Furthermore, pre-incubationwith p38 MAPK inhibitors (Dilmapimod and PH 797804) was shown to dosedependently inhibit the induction of HSP27 phosphorylation by the A549viral soup, confirming that this induction is a p38MAPK dependentprocess within these immune cells (FIG. 8).

The effect of A549 viral soup on inflammatory cytokine production inimmune cells as measured by electrogenerated chemiluminescence (seeabove) was also explored. As shown in FIG. 8, A549 viral soup was foundto induce the production of key inflammatory cytokines (TNFα, IL-1-β,IL-6 and CXCL8) to a greater extent compared with control A549uninfected soup. Pre-incubation of p38 MAPK inhibitors (Dilmapimod andPH 797804) on immune cells prior to A549 viral soup application wasfound to significantly attenuate the

A549 viral soup induced release of TNFα, IL-1-β, IL-6 and CXCL8 (FIG.8). These data demonstrate that the release of inflammatory mediators inresponse to A549 viral soup application in immune cells is a p38MAPKdriven process.

Additional ex vivo data were generated from immune cells whereby theinduction of inflammatory mediators in response to A549 viral soup wascompared to known inflammatory stimulants (anti-CD3 and LPS). Theinduction of TNFα, IL-1-β, IL-6 and IL-8 by A549 viral soup was found tobe greater compared with these known inflammatory stimulants (FIG. 9).

Example 3 p38 MAPK Inhibitory Activity

The enzyme inhibitory activity of a compound may be determined byfluorescence resonance energy transfer (FRET) using synthetic peptideslabelled with both donor and acceptor fluorophores (Z-LYTE, Invitrogen).

Recombinant, phosphorylated p38 MAPK gamma (MAPK12:Millipore) is dilutedin HEPES buffer, mixed with the candidate compound at desired finalconcentrations and incubated for two hours at room temperature. The FRETpeptide (2 μM) and ATP (100 μM) are next added to the enzyme/compoundmixture and incubated for one hour. Development reagent (protease) isadded for one hour prior to detection in a fluorescence microplatereader. The site-specific protease only cleaves non-phosphorylatedpeptide and eliminates the FRET signal. Phosphorylation levels of eachreaction are calculated using the ratio of coumarin emission (donor)over fluorescein emission (acceptor) with high ratios indicating highphosphorylation and low ratios, low phosphorylation levels. Thepercentage inhibition of each reaction is calculated relative tonon-inhibited control, and the 50% inhibitory concentration (IC₅₀ value)then calculated from the concentration-response curve.

For p38 MAPK alpha (MAPK14: Invitrogen), enzyme activity is evaluatedindirectly by determining activation/phosphorylation of the down-streammolecule, MAPKAP-K2. The p38 MAPK a protein is mixed with its inactivetarget MAPKAP-K2 (Invitrogen) and the candidate compound for two hoursat room temperature. The FRET peptide (2 μM), which is a phosphorylationtarget for MAPKAP-K2, and ATP (10 μM) are then added to theenzymes/compound mixture and incubated for one hour. Development reagentis then added and the mixture incubated for one hour before detection byfluorescence completed the assay protocol.

Example 4 P38MAPK Inhibition (p38i) Versus Inhibition of Other PotentialTargets

As indicated in Example 1 above, a number of targetable nodes in the 95pathway routes highlighted by transcriptomic and bioinformatics wereidentified. The compound profiling experiments in Example 2 show thatp38i is effective in reducing the production of inflammatory mediatorrelease in cell types relevant to the pathology of severe influenza.This was found not to be the case for 9 other nodes that were examined:PI3K, MEK, ERK, JNK, JAK/STAT, PKC, SRC, BtK and mTor. Drug inhibitionof none of these 9 nodes gave an inhibition profile as effective as p38iin epithelial, endothelial and immune cells.

By way of example, data comparing p38i versus mitogen-activated proteinkinase (MEK) inhibition (MEKi) by MEK inhibitors Refametinib (Iverson Cet al., RDEA119/BAY 869766: a potent, selective, allosteric inhibitor ofMEK1/2 for the treatment of cancer. Cancer Res., 2009; 69: 6839-6847)and Selumetinib (Huynh H et al., Targeted inhibition of theextracellular signal-regulated kinase pathway with AZD6244 (ARRY-142886)in the treatment of hepatocellular carcinoma, Molecular CancerTherapeutics, 2007; 6:138-146) are presented in FIG. 10 of theaccompanying drawings.

Neither Refametinib nor Selumetinib showed dose-dependent inhibition ofIP10 production in endothelial cells stimulated with HBEC viral soup andactually appeared to increase levels of IP10 at higher drugconcentrations (see FIG. 10).

A number of potential drug targets have been proposed for severeinfluenza (e.g. Liu Q et al., 2015 and Fedson DS, 2009). p38i was alsocompared versus drug compounds for a selection of these proposedtargets.

For these experiments, PH797804 was benchmarked versus corticosteroid(methyl prednisolone), macrolide (Azithromycin), PPAR agonist(Pioglitazone), PDE4 inhibitor (Roflumilast), NFκB inhibitor (EVP4593)and statin (Pravastatin) at four drug concentrations (1 nM, 10 nM, 100nM and 1000 nM) in endothelial cells (HUVECs stimulated with HBEC viralsoup), or immune cells (PBMCs plus granulocytes stimulated with A549viral soup as described in Example 2 above).

The effects of drug compound administration on IP-10, IL-8 and MCP-1production from endothelial cells and on IL-1β, IL-6, IL-8 and TNF-αproduction from immune cells was assayed using electrogeneratedchemiluminescence. In immune cells, corticosteroid and macrolide drugtreatment showed dose-dependent inhibition of all four assayedcytokines, whereas p38i showed dose-dependent inhibition of only threeof the four. The inhibitory profile of the other drugs tested wasvariable and did not match that of corticosteroid, macrolide, or p38i.The results are summarised in Table 5 below.

TABLE 5 Comparison of inhibitory effects of drug compounds forliterature-proposed targets for severe influenza versus p38i. PBMCs plusgranulocytes were isolated as described in Example 2 and stimulated withA549 viral soup. Secreted cytokine levels were assayed byelectrogenerated chemiluminescence and IC₅₀ and iMax values werecalculated from the dose responses using non-linear regression fit usinga scientific 2D graphing and statistics software package (GraphPadPRISM ® version 6.07 software). Where data did not show a dose-dependentinhibition, the IC₅₀ and iMax values were not calculated (empty boxes).Corticosteroid NFkB inhibitor p38 inhibitor Methyl Macrolide PPARagonist PDE4 inhibitor QNZ Statin PH797804 Prednisolone AzithromycinPioglitazone Roflumilast (EVP4593) Pravastatin IC₅₀ iMax IC₅₀ iMax IC₅₀iMax IC₅₀ iMax IC₅₀ iMax IC₅₀ iMax IC₅₀ iMax (nM) (%) (nM) (%) (nM) (%)(nM) (%) (nM) (%) (nM) (%) (nM) (%) IL1B 9.7 79% 8.4 77% 0.5 50% IL8 2.387% 8.4 90% 0.8 46% 0.1 37% 0.09 31% 0.2 47% TNFa 3.9 91% 20.2 79% 86.963% 10.8 74% 43.6 37% IL6 36.6 57% 119 45% 0.02 41%

The inhibition plots for IL1-b and TNFa for the compounds tested areshown in FIGS. 11 and 12.

With endothelial cells, in contrast to immune cells, only p38 and NFKBinhibitors showed dose-dependent inhibition of the three cytokinesassayed. None of the other drugs tested showed a comparable inhibitoryeffect. The results are summarised in Table 6 below.

TABLE 6 Comparison of inhibitory effects of drug compounds forliterature-proposed targets for severe influenza versus p38i. HUVECcells were stimulated with HBEC viral soup. Secreted cytokine levelswere assayed by electrogenerated chemiluminescence and IC₅₀ and iMaxvalues were calculated from the dose responses using non-linearregression fit with a scientific 2D graphing and statistics softwarepackage (GraphPad PRISM ® version 6.07 software). Where data did notshow a dose-dependent inhibition, the IC₅₀ and iMax values were notcalculated. Corticosteroid NFkB inhibitor p38 inhibitor Methyl MacrolidePPAR agonist PDE4 inhibitor QNZ Statin PH797804 PrednisoloneAzithromycin Pioglitazone Roflumilast (EVP4593) Pravastatin IC₅₀ iMaxIC₅₀ iMax IC₅₀ iMax IC₅₀ iMax IC₅₀ iMax IC₅₀ iMax IC₅₀ iMax (nM) (%)(nM) (%) (nM) (%) (nM) (%) (nM) (%) (nM) (%) (nM) (%) IP10 7.5 88% 3.372% 78 35% IL8 28.9 86% 6.5 60% MCP1 13.1 36% 3.84 44%

The inhibition plots for IP10 and IL8 are shown in FIGS. 13 and 14.

Based on the results obtained in the immune cell experiments, thesuperiority of p3 8i versus the other drugs was unexpected, especiallymethyl prednisolone, which is used routinely in clinical settings totreat a range of inflammatory diseases (e.g. asthma) and is commonlyprescribed for severe influenza, although there is uncertainty overtheir potential benefit or harm (Rodrigo C et al., Corticosteroids asadjunctive therapy in the treatment of influenza, Cochrane Database ofSystematic Reviews, 2016, Issue 3. Art. No.: CD010406. DOI:10.1002/14651858.CD010406.pub2]. NFκB is downstream of p38, so theinhibition profile seen is not unexpected.

Example 5 Levels of a Number of Cytokines in Serum from PatientsHospitalised for Severe Influenza Were Significantly Raised Relative toInfluenza-Infected Individuals Without Severe Influenza

Cytokine levels in serum samples from 30 subjects hospitalised withsevere influenza during the 2015 influenza season and from 28 healthysubjects infected after intranasal inoculation with influenza A/H3N2Perth/16/2009 virus were assayed using electrogeneratedchemiluminescence. In the case of the former, a serum sample preparedfrom a blood sample collected 24-72 hours after the subject was admittedto hospital was analysed. In the case of the latter, serum was analysedfrom blood samples collected at 12 pre-determined intervals (Day −1 toDay 28). Eight cytokines were observed to be significantly raised in thehospitalised versus the infected healthy subjects: IL-8, IL-7, IL-16,Eotaxin, IP10, MCP1, MCP4 and VEGF. The results for four of these areshown in FIG. 15.

The results show that the hospitalised subjects are distinguishable fromthe healthy infected subjects in terms of their serum cytokine profiles.

Example 6 Effects of p38 MAPK Inhibition with the Compound of FormulaIII on Inflammatory Mediator Release in Key Cell Types Relevant toSevere or Persistent Influenza

The p38 MAPK inhibitor of Formula III was used in in vitro and ex vivoexperiments (Example 8 of WO 2004/076450 A1 (J. Uriach Y CompañiaS.A.)).

Experimental testing of p38 MAPK inhibition by the inhibitor of FormulaIII was carried out in three cell types that are key players in thepathology of severe and persistent influenza: epithelial; endothelial;and immune cells (FIG. 4). In these experiments, cells were pre-treatedwith simple and/or complex stimuli in order to simulate the action ofinflammatory mediators that are produced from influenza-infectedepithelial cells. In the case of the former, tumour necrosis factoralpha (TNFa) plus interleukin 6 (IL-6) was used to stimulate endothelialand immune cells. In the case of the latter, conditioned medium (viral“soup”) derived either from influenza virus infected A549 cells(adenocarcinoma human alveolar basal epithelial cells), or primary humanbronchial epithelial cells (HBECs) was used to stimulate epithelial,endothelial and immune cells.

Epithelial Cells

For the production of conditioned medium (viral soup), A549 cells orHBECs were infected with high titre stocks of Influenza A/Perth/16/2009(H3N2) virus. Viral stocks were produced by infection of Madin-DarbyCanine Kidney [MDCK cells, available from the American Type CultureCollection as MDCK (NBL-2) (ATCC® CCL-34™)]. MDCK cells were cultured to85-90% confluence in Minimal Essential Medium (MEM) containing 10%foetal bovine serum. Cells were washed twice with infection medium(Advanced Dulbecco's Modified Eagle Medium [DMEM] containing 1.06 USP/NFunits per ml TPCK trypsin). The cells were infected with InfluenzaA/Perth/16/2009 (H3N2) virus stock at 0.01 MOI for one hour in infectionmedium. At the end of the incubation period, unbound virus particleswere removed from the cells by washing once with infection medium. Thecells were then overlaid with fresh infection medium and incubated for48 hours at 37° C. in 5% CO₂/air. After incubation, the flasks of cellswere frozen at −80° C. for 24 hours, thawed at room temperature andvirus supernatants were centrifuged (2000 g, 10 min) before poolingtogether, aliquoting and storing at −80° C.

The viral stocks prepared were used for the generation of viral soups.Viral growth kinetics were optimised by determining multistep growthcurves. A549 cells or HBECs were infected with virus at an MOI of 0.01at 37° C. for one hour. Following incubation, the cells were washed andoverlaid with infection medium. The samples were harvested at varioustime points for 72 hours for viral titre determination by TCID5o assayon MDCK cells and the presence of inflammatory mediators was assessed byMSD chemiluminescence assay using methods as recommended by the vendor(https://web.archive.org/web/20160522190937/https://www.mesoscale.com/).

For the preparation of A549 viral soup, cells were cultured in MEM plus10% foetal bovine serum to 85-90% confluence. The cells were washedtwice with infection medium then infected with Influenza A/Perth/16/2009(H3N2) virus stock at 0.01 MOI for one hour in infection medium. Unboundvirus was removed from the cells at the end of the incubation period bywashing once with infection medium before overlaying with infectionmedium. The cells were incubated for 48 hours at 37° C. in 5% CO₂/air.After incubation, the viral soup was collected from the culture flasks,centrifuged (2000 g, 10 min) and pooled together. The viral soup wasaliquoted and stored at −80° C. For the preparation of HBEC viral soupcells were cultured to passage P-3 or P-4 in Bronchial Epithelial CellGrowth Medium (BECGM; Lonza). For infection, cells were washed twicewith BECGM then infected with Influenza A/Perth/16/2009 (H3N2) virusstock at 0.01 MOI for one hour in infection medium (BECGM containing1.06 USP/NF units per ml TPCK trypsin). The cells were then overlaidwith infection medium and incubated for 48 hours at 37° C. in 5%CO₂/air. The HBEC viral soup was then processed in a similar way to A549soup.

Prior to experimental testing, levels of pro-inflammatory mediators inboth A549 and HBEC soup preparations were measured by MSDchemiluminescence analysis. Both soup preparations were found to containelevated levels of pro-inflammatory cytokines (IL1-β, IL-6, IL-8, IL-10,TNFα and RANTES; FIG. 5) and demonstrated to induce p38MAPK signallingby demonstration of phosphorylation of both p38MAPK and phosphorylationof the downstream signalling protein HSP27 by western blot analysis(FIG. 5). For western blotting, viral soup-treated cells were washedwith PBS and lysed in RIPA buffer with protease inhibitor (Sigma-P8340),phosphatase inhibitor cocktails 2 and 3 (Sigma P5726 and P0044) andphosphatase inhibitors sodium orthovanadate and sodium fluoride (both,www.sigmaaldrich.com) on ice. Protein concentrations were determinedusing the Pierce BCA Protein Assay Kit (www.thermofisher.com).Equivalent concentrations of each sample in 4×Laemmli sample buffer(BIO-RAD 161-0747) with 2-mercaptoethanol were analysed on a 12%polyacrylamide gel in Tris-glycine-SDS buffer and then transferred tonitrocellulose membrane. Membranes were blocked using 5% milk powder,then hybridised with p38 MAPK rabbit antibody (Cell SignallingTechnologies, cat. no. 9212S) and phosphorylated p38 MAPK rabbitantibody (Cell Signalling Technologies, cat. no. 9211S), or HSP27antibody (Cell signalling Technologies, cat. no. 2402) and phospho-HSP27antibody (Cell signalling Technologies, cat. no. 9709). Secondaryantibodies were anti-rabbit HRP (Cell signalling Technologies catalogueno. CS7074P2) or anti-mouse HRP (Cell signalling, catalogue no. 7076S),respectively. Membranes were stripped for re-probing using Restore™ PLUSWestern Blot Stripping Buffer (Life technologies # 46430). The membraneswere treated with Amersham ECL Prime Western Blotting Detection Reagent(GE/Amersham #RPN2232) and imaged using ChemiDoc™ Touch Imaging System(BIO-RAD). Analysis was performed using Image Lab software.

The effect of HBEC viral soup on inflammatory mediator production inHBEC cells was measured by MSD analysis. As shown in FIG. 16, HBEC soupinduced the production of key inflammatory cytokines as exemplified byIL-6 and IP-10. Treatment of HBECs with the p38 MAPK inhibitor ofFormula III prior to HBEC viral soup application attenuated release ofboth cytokines (FIG. 16).

Endothelial Cells

Both simple (TNFa plus IL-6) and complex (HBEC viral soup) stimuli wereapplied to Human Umbilical Vein Endothelial Cells (HUVECs) to simulatethe interaction of inflammatory mediators produced by influenza-infectedepithelial cells with endothelial cells.

Inflammatory cytokine production by HUVEC cells treated with either TNFaplus IL-6, or HBEC viral soup as measured by MSD analysis was explored.As shown in FIG. 17, both stimuli induced the production of keyinflammatory cytokines as exemplified by IL-8 and IP-10. However,treatment of HUVEC cells with p38 MAPK inhibitor of Formula III prior tostimulation was shown to attenuate this (FIG. 17).

Immune Cells

Both simple (TNFa plus IL-6) and complex (A549 viral soup) stimuli wereapplied to isolated human Peripheral Blood Mononuclear Cells (PBMCs) tosimulate the interaction of inflammatory mediators that are producedfrom influenza-infected epithelial cells on immune cells. PBMCs wereisolated from human blood according to the manufacturers recommendations(Boyum, A., Separation of leucocytes from blood and bone marrow, Scand.J. Clin. Lab. Invest., 1968, 21, suppl. 97).

Inflammatory cytokine production in immune cells treated with eitherTNFa plus IL-6, or A549 viral soup as measured by MSD analysis wasexplored. As shown in FIG. 18, both stimuli induced the production ofkey inflammatory cytokines as exemplified by TNFα and

IL-8. However, treatment of PBMCs with the p38 MAPK inhibitor of FormulaIII prior to stimulation was found to attenuate this (FIG. 18).

Example 7 Combination of the p38 MAPK Inhibitors with the Antiviral DrugOseltamivir

Successful therapy of severe influenza is envisaged to depend oneffectively targeting both the viral infection phase (phase 1) viaantiviral drugs and the later (post-infection) inflammatory phase (phase2) with immunomodulatory drugs. As antiviral treatment is recommended asearly as possible for any patient with confirmed or suspected influenzawho: is hospitalised; has severe, complicated, or progressive illness;or is at higher risk for influenza complications[https://www.cdc.gov/flu/professionals/antivirals/summary-clinicians.htm],it is important that an immunomodulator does not impede the action ofthe antiviral drug as both will likely be given at the same time. Toexamine this possibility the effect of combining oseltamivir with eitherof two p38MAPK inhibitors (PH797804 and Dilmapimod) was investigated byexamining the effect that these two drugs had on the ability ofoseltamivir to suppress viral infection in HBECs.

HBECs were infected with Influenza A/Perth/16/2009 (H3N2) virus stock at0.01 MOI and the effect of oseltamivir in combination with eitherPH797804 or Dilmapimod on viral infection was examined. The oseltamivirused in this Example was in the form of oseltamivir carboxylate, whichis the active metabolite of oseltamivir and pharmaceutically acceptablesalts thereof (e.g. it is the active metabolite of oseltamivirphosphate). Viral titre was determined by TCID₅₀ assay [WHO manual forthe laboratory diagnosis and virological surveillance of influenza;http://whqlibdoc.who.int/publications/2011/9789241548090_eng.pdf].Whereas oseltamivir treatment reduced TCID5o, neither PH797804 norDilmapimod treatment showed this effect. When oseltamivir was combinedwith either PH797804 or Dilmapimod, TCID₅₀ was reduced to the level seenwith oseltamivir on its own, indicating that p38MAPK inhibitor therapycould be used in combination with oseltamivir without impacting itsantiviral activity (FIG. 19).

In a clinical setting it is envisaged that reduction of viral load(phase 1) by antiviral therapy would be monitored by measuring viral RNAlevels using quantitative reverse transcriptase polymerase chainreaction (qRT-PCR), and effects of p38 inhibition on inflammation (phase2) would be monitored by measuring the levels of inflammatory mediatorssuch as those seen in patients with severe influenza (example 5) by, forexample, MSD analysis of serum samples.

Example 8 Effects of p38 MAPK Inhibition with the Compound of Formula IIon Inflammatory Mediator Release in Key Cell Types Relevant to Severe orPersistent Influenza

The p38 MAPK inhibitor of Formula II was used in in vitro and ex vivoexperiments (Example 18 of WO 2004/076450 Al (J. Uriach Y Compañia S.A.)). As described above, the chemical structure of this compound is asfollows:

Experimental testing of p38 MAPK inhibition by the inhibitor of FormulaII was carried out in endothelial and immune cells. In theseexperiments, cells were pre-treated with TNFa plus IL-6, or viral soupsprepared as described above in [00190] to [00193].

Endothelial Cells

Inflammatory cytokine production by HUVEC cells treated with either TNFaplus IL-6, or HBEC viral soup was measured by MSD analysis. As shown inFIG. 20, both stimuli induced the production of key inflammatorycytokines as exemplified by IL-8 and IP -10. However, treatment of HUVECcells with p38 MAPK inhibitor of Formula II prior to stimulation wasshown to attenuate this (FIG. 20).

Immune Cells

Both simple (TNFa plus IL-6) and complex (A549 virus soup) stimuli wereapplied to isolated human Peripheral Blood Mononuclear Cells (PBMCs) tosimulate the interaction of inflammatory mediators that are producedfrom influenza-infected epithelial cells on immune cells. PBMCs wereisolated from human blood according to the manufacturer'srecommendations (Boyum, A., Separation of leucocytes from blood and bonemarrow, Scand. J. Clin. Lab. Invest., 1968, 21, suppl. 97).

Inflammatory cytokine production in immune cells treated with eitherTNFa plus IL-6, or A549 virus soup as measured by MSD analysis wasexplored. As shown in FIG. 21, both stimuli induced the production ofkey inflammatory cytokines as exemplified by MIP-la and IL-8 (TNFa plusIL-6 as stimulus) and TNFa and IL-8 (A549 virus soup as stimulus).However, treatment of PBMCs with the p38 MAPK inhibitor of Formula IIprior to stimulation was found to attenuate this (FIG. 21).

Example 9 Combination of Compound with Formula II with the AntiviralDrug Oseltamivir

As it is important that an immunomodulator does not impede the action ofantiviral drugs that are likely to be given at the same time, the effectof combining compound of Formula II with oseltamivir was investigated byexamining the effect that this drug compound has on the ability ofoseltamivir to suppress viral infection in HBECs. Conversely, the effectthat this combination might have on the anti-inflammatory properties ofcompound with Formula II was also examined.

HBECs were infected with Influenza A/Perth/16/2009 (H3N2) virus stock at0.01 MOI and the effect of oseltamivir in combination with compound ofFormula II on viral infection was examined. The oseltamivir used in thisExample was in the form of oseltamivir carboxylate, which is the activemetabolite of oseltamivir and pharmaceutically acceptable salts thereof(e.g. it is the active metabolite of oseltamivir phosphate).Viral titrewas determined by TCID50 assay [WHO manual for the laboratory diagnosisand virological surveillance of influenza;http:/lwhqlibdoc.who.int/publications/2011/9789241548090 eng.pdf].Whereas oseltamivir treatment alone reduced TCID₅₀, treatment with thecompound of Formula II alone did not significantly reduce the TCID₅₀.When oseltamivir was combined with compound of Formula II, TCID₅₀ wasreduced to the level seen with oseltamivir on its own, indicating thatp38MAPK inhibitor therapy could be used in combination with oseltamivirwithout impacting oseltamivir's antiviral activity (FIG. 22).Conversely, oseltamivir alone had no observable effect on theanti-inflammatory properties of the compound of Formula II (FIG. 23).

In a clinical setting it is envisaged that reduction of viral load byantiviral therapy would be monitored by measuring viral RNA levels usingquantitative reverse transcriptase polymerase chain reaction (qRT-PCR),and the effects of p38 inhibition on inflammation would be monitored bymeasuring the levels of inflammatory mediators such as those seen inpatients with severe influenza (Example 5) by, for example, MSD analysisof serum samples.

Abbreviations

ATP Adenosine triphosphate

A549 Adenocarcinoma human alveolar basal epithelial cells

BECGM Bronchial epithelial cell growth medium

Btk Bruton's tyrosine kinase

CXCL8 Interleukin 8

CD3 Cluster of differentiation protein 3

DMEM Dulbecco's modified eagle medium

ERK Extracellular signal-regulated kinases)

FRET Fluorescence resonance energy transfer

GSK Glaxo Smith-Kline

HBEC Human bronchial epithelial cells

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HSP27 Heat shock protein 27

HUVEC Human vascular endothelial cells

IC₅₀ Half maximal inhibitory concentration

iMax Maximal inhibition (as a %)

IL1-b Interleukin 1 beta

IL-6 Interleukin 6

IL-8 Interleukin 8

IPA Ingenuity Pathways Analysis

IP-10 Interferon gamma-induced protein 10

JAK/STAT Janus kinase/signal transducer and activator of transcription

JNK c-Jun N-terminal kinase

LPS Lipopolysaccharide

MAPKAP-K2 MAP kinase-activated protein kinase 2

MOI Multiplicity of infection

MCP-1 Monocyte chemotactic protein-1

MDCK Madin Darby canine kidney

MEK Mitogen-activated protein kinase kinase

MEKi MEK inhibition (by drug)

MEM Minimal essential medium

mTOR Mechanistic target of rapamycin

MSD Mesoscale Discovery

NFκB Nuclear factor kappa-light-chain-enhancer of activated B cells

P38 MAPK P38 Mitogen-activated protein kinases

P38i p38 inhibition (by drug)

PBMC Peripheral blood mononuclear cells

PBS Phosphate buffered saline

PDE4 Phosphodiesterase 4

PKC Protein kinase

PPAR Peroxisome proliferation-activated receptor

RANTES Regulated on activation, normal T cell expressed and secreted

RIPA Radio immuno precipitation assay

SDS Sodium dodecyl sulphate

SRC Src kinase

TCID₅₀ Tissue culture infective dose

TNFa/TNFα Tumour necrosis factor alpha

TPCK Tosyl phenylalanyl chloromethyl ketone

USP/NF United States pharmacopeia and the national formula

1-74. (canceled)
 75. A method of treating or preventing hypercytokinemiain a human or animal patient in need thereof comprising administering tothe patient a therapeutically or prophylactically effective amount of ap38 MAP kinase inhibitor of Formula I, or a pharmaceutically acceptablesalt or solvate thereof:

wherein R is C₁₋₃alkyl, optionally substituted by one or more halo,NR¹R² or hydroxy, and R¹ and R² are independently H, halo or C₁₋₃alkyl,optionally substituted by one or more F.
 76. The method of claim 75,wherein the p38 MAP kinase inhibitor is of Formula II, or apharmaceutically acceptable salt or solvate thereof:


77. The method of claim 75, wherein the p38 MAP kinase inhibitor is ofFormula III, or a pharmaceutically acceptable salt or solvate thereof:


78. The method of claim 75, wherein the hypercytokinemia occurs due toone or more of severe influenza virus infection; graft-versus-hostdisease (GVHD); acute respiratory distress syndrome (ARDS); sepsis;Ebola; smallpox; systemic inflammatory response syndrome (SIRS);bacterial infection; and cancer.
 79. The method of claim 75, wherein thehypercytokinemia occurs due to severe influenza virus infection.
 80. Themethod of claim 75, further comprising administering an antimicrobialagent.
 81. (canceled)
 82. The method of claim 80, wherein theantimicrobial agent is an antiviral agent.
 83. The method of claim 82,wherein the antiviral agent is oseltamivir or a pharmaceuticallyacceptable salt thereof.
 84. The method of claim 75, further comprisingadministering an anticancer agent.
 85. (canceled)
 86. The method ofclaim 75, wherein the p38 MAP kinase inhibitor is administered orally.87. (canceled)
 88. (canceled)
 89. A method of treating or preventinghypercytokinemia in a human or animal patient in need thereof comprisingadministering to the patient a therapeutically or prophylacticallyeffective amount of a p38 MAP kinase inhibitor.
 90. The method of claim89, which further comprises administering an antimicrobial agent. 91.The method of claim 90, wherein the antimicrobial agent is an antiviralagent.
 92. The method of claim 91, wherein the antiviral agent isoseltamivir or a pharmaceutically acceptable salt thereof.
 93. Themethod of claim 89, which further comprises administering to the patienta therapeutically or prophylactically effective amount of an anticanceragent.
 94. The method of claim 89, wherein the p38 MAP kinase inhibitoris administered orally.